Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
  • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
  • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
  • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
  • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
  • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
  • A61K39/145—Orthomyxoviridae, e.g. influenza virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
  • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/51—Nanocapsules; Nanoparticles
  • A61K9/5107—Excipients; Inactive ingredients
  • A61K9/5123—Organic compounds, e.g. fats, sugars
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
  • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
  • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
  • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
  • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
  • C07C229/16—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C233/00—Carboxylic acid amides
  • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
  • C07C233/16—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
  • C07C233/17—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
  • C07C233/18—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
  • C07C271/06—Esters of carbamic acids
  • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
  • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
  • C07C271/16—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by singly-bound oxygen atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6018—Lipids, e.g. in lipopeptides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S977/00—Nanotechnology
  • Y10S977/70—Nanostructure
  • Y10S977/788—Of specified organic or carbon-based composition
  • Y10S977/797—Lipid particle

Definitions

• the present disclosure relates to novel lipidic compounds which can be used to form lipid nanoparticles for delivery of therapeutic agents, such as nucleic acid, for instance in combination with other lipids, such as neutral lipids, steroids and polymer conjugated lipids.
• therapeutic agents such as nucleic acid
• other lipids such as neutral lipids, steroids and polymer conjugated lipids.
• the formulations prepared with the lipidic compounds as described herein enable to induce an immune response after administration of antigen-coding nucleic acid.
• the polynucleotide therapeutics field has seen remarkable progress over the recent years.
• Polynucleotides include various nucleic acids-based compounds such as messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, or immune stimulating nucleic acids.
• mRNA messenger RNA
• antisense oligonucleotides ribozymes
• DNAzymes DNAzymes
• plasmids or immune stimulating nucleic acids.
• Some nucleic acids such as mRNA, plasmids and ssDNA, can be used to induce the expression of specific cellular products useful in the treatment of, for example, diseases related to a deficiency of a protein or enzyme, or for the expression of a vaccine antigen to induce specific immune responses.
• the therapeutic applications of translatable nucleotide delivery are extremely broad as constructs can be synthesized to produce any chosen protein sequence, whether or not indigenous to the system.
• the expression products of the nucleic acid can augment existing levels of protein, replace missing or non-functional versions of a protein, introduce new protein and associate functionality in a cell or organism or expose to a foreign protein in order to induce a specific immune response.
• challenges associated with the delivery of polynucleotides to affect a desired response in a biological system and the effective delivery of polynucleotides to their intracellular sites of action remains a major issue.
• the polynucleotides To be efficiently delivered to their site of action, the polynucleotides must be (i) protected from enzymatic and non-enzymatic degradation, (ii) appropriately distributed in the biologic compartment of interest, (iii) effectively and efficiently internalized by the targeted cells, and then (iv) delivered to the intracellular compartment where the relevant translation machinery resides.
• Lipid nanoparticles formed from cationic lipids formulated with other lipid components, such as neutral lipids, cholesterol, and PEGylated lipids have been used to protect the polynucleotide from degradation and facilitate its cellular uptake.
• lipid nanoparticles-based vehicles that comprise a cationic lipid component have shown promising results with regards to encapsulation, stability and site localization, there remains a great need for improvement of lipid nanoparticles-based delivery systems. Indeed, many of the cationic lipids that are employed to construct such lipid nanoparticles may be toxic to the targeted cells, and accordingly may be of limited use, notably in quantities necessary to successfully deliver encapsulated materials to such target cells. Therefore, there remains a need for improved lipid nanoparticles that demonstrate improved pharmacokinetic properties, and which are capable of delivering various types of nucleic acids to a wide variety cell types and tissues with enhanced efficiency.
• neutral or negatively lipid nanoparticles In contrast to positively charged lipid nanoparticles, neutral or negatively lipid nanoparticles have generally relatively improved pharmacokinetic properties. However, they usually yield low encapsulation efficiency. Therefore, there remains a need for novel lipids able to combine the high efficiency of polynucleotides encapsulation rate associated with cationic lipid and the pharmacokinetic properties of neutral or lowly charged lipid nanoparticles. In prior art, this has been achieved with ionizable cationic lipids displaying a cationic charge at low pH and a neutral charge at neutral pH. However, such ionizable lipids may display some toxic effects, either locally, systemically or both, upon in vivo administration.
• lipidic compounds having reduced toxicity and are capable of efficiently encapsulating polynucleotides and delivering encapsulated polynucleotides to targeted cells, tissues and organs.
• Improved lipids and lipid nanoparticles for the delivery of polynucleotides would also provide optimal polynucleotide(s)/lipid(s) ratios, protect the polynucleotides from degradation and clearance in serum, be suitable for systemic or local delivery, and provide intracellular delivery of the polynucleotide.
• lipid- polynucleotide particles should be well-tolerated and provide an adequate therapeutic index, such that patient treatment at an effective dose of the polynucleotide is not associated with unacceptable toxicity and/or risk to the patient.
• one of the objects of the present disclosure relates to new cleavable lipidic compounds comprising at least one terminal radical of formula (I): Y-(CHR) n -Z-(CHR’) p -Q * (I) wherein: - * is the end linked, directly or not, to one C 10 to C 60 and preferably to C 10 to C 55 lipophilic or hydrophobic tail-group, - Y is a radical selected in the group consisting of a C 1 -C 5 alkyl, a C 1 - C 5 alkoxy, a C 1 -C 5 acyl, a C 1 -C 5 hydroxyalkyl, a C 1 -C 5 aminoalkyl, a C 1 -C 5 alkylcarboxyl ester, an acetamido, a N,N-C 1 -C 5 alkylamido, a C 1 -C 5 fluoroalkyl, for example a C 1
• the compound as disclosed herein is a compound of formula (II) Y-(CHR) n -Z-(CHR’) p -Q-A- R 1 (II) wherein: - Y, R, n, Z, R’, p and Q are as defined herein; - R 1 is a C 10 to C 60 and preferably to C 10 to C 55 lipophilic or hydrophobic tail-group; and - A is a spacer arm having from 2 to 24, for example from 2 to 18, for example from 4 to 12 carbon atoms, or for example from 2 to 12 carbon atoms, in a branched or unbranched linear, saturated or unsaturated hydrocarbon chain, said chain being interrupted by one or several atoms of oxygen and/or moieties selected among — S—S— ; (C ⁇ O)— O—; —O—(O ⁇ C)— ;—(C ⁇ O)—NH—; —NH—(C ⁇ O)— O—;—S—;
• the compound according to the disclosure is a compound of formula (IIa) Y-(CHR) n -NH-CH 2 -CO-O-(CHR’) p -NH-CH 2 -CO-O-A-R 1 (IIa) wherein: - Y, R, n, R’ and p are as previously defined; - R 1 is a C 10 to C 60 and preferably to C 10 to C 55 lipophilic or hydrophobic tail-group; and - A is a spacer arm as previously defined; or one of the pharmaceutically acceptable salts of said compound of formula (II); and any of its racemic, enantiomeric and diastereoisomeric isomer forms.
• the disclosure relates to a method for manufacturing lipid nanoparticles containing a nucleic acid, wherein the method comprises at least the steps of: a) solubilizing, in a water miscible organic solvent, at least one lipidic compound as described herein, b) mixing the organic solvent obtained at step a) with at an aqueous solvent buffered at a pH ranging from about 3.0 to about 4.5 and comprising at least one nucleic acid, and c) obtaining said nucleic acid containing lipid nanoparticles in the aqueous solvent.
• the lipid nanoparticles manufacturing method as described herein further comprises a step d) of increasing the pH of the aqueous solvent containing the lipid nanoparticles obtained at step c) at a pH ranging about 5.0 to about 8.5, for example from about 5.5 to about 8.0, for example from about 6.0 to about 7.5, and for example from about 6.5 to about 7.0.
• the disclosure relates to lipid nanoparticles obtainable according the manufacturing method as disclosed herein.
• the disclosure relates to a method for manufacturing a pharmaceutical composition comprising at least the steps of: i) manufacturing at least one lipid nanoparticle according to the method as described herein, and ii) combining the lipid nanoparticles obtained at step i) with at least one pharmaceutically acceptable excipient or carrier.
• the disclosure relates to a method for manufacturing an immunogenic composition comprising at least the steps of: i) manufacturing at least one lipid nanoparticle according to the method as described herein, said lipid nanoparticle containing at least one nucleic acid encoding for at least one antigen, and ii) combining the lipid nanoparticles obtained at step i) with at least one pharmaceutically acceptable excipient or carrier.
• the disclosure relates to lipid nanoparticles obtainable according to a method as disclosed herein.
• the disclosure relates to a lipid nanoparticle comprising at least one lipidic compound of formula (IV): HO-L (IV) wherein L is a lipophilic or hydrophobic tail-group as described herein, for instance a C 10 to C 55 lipophilic or hydrophobic tail-group, and at least one nucleic acid.
• the disclosure relates to a pharmaceutical composition comprising at least one lipid nanoparticle as described herein, and at least one pharmaceutically acceptable excipient or carrier.
• the disclosure relates to an immunogenic composition comprising at least one lipid nanoparticle as disclosed herein wherein the least one nucleic acid encodes for at least one antigen.
• the disclosure relates to a composition comprising at least one lipid nanoparticle as disclosed herein as a medicament.
• the disclosure relates to a composition comprising at least one lipid nanoparticle as disclosed herein, for use in a therapeutic method for preventing and/or treating a disease selected in a group consisting of infectious diseases, allergies, autoimmune diseases, rare blood disorders, rare metabolic diseases, rare neurologic diseases, and tumour or cancer diseases.
• the disclosure relates to a composition comprising at least one lipid nanoparticle as disclosed herein for use as an immunogenic composition.
• the disclosure also relates to a method of preventing and/or treating a disease in an individual in need thereof, wherein the method comprises administering an effective amount of at least one lipid nanoparticle as disclosed herein, to said individual.
• a method as disclosed herein may be for preventing and/or treating infectious diseases, allergies, autoimmune diseases, rare blood disorders, rare metabolic diseases, rare neurologic diseases, and tumor or cancer diseases.
• the disclosure also relates to a use of at least one lipid nanoparticle as disclosed herein for the manufacture of a medicament for preventing and/or treating infectious diseases, allergies, autoimmune diseases, rare blood disorders, rare metabolic diseases, rare neurologic diseases, and tumor or cancer diseases.
• Figure 1 Hemagglutination inhibiting antibody mean titers (HI titers) measured in serum from mice post-2 immunization either with LNPs L319 or with LNPs Lip.
• FIG. 2 Bioluminescence signal acquisition monitoring protein expression in injected site (quadriceps) following intramuscular administration of LNPs 319 or LNPs Lip.
• III loaded with 5 ⁇ g of mRNA encoding Luciferase (mRNA-Luc) in female BALB/c ByJ mice. The luminescence level was evaluated by an ROI applied to the injection site zone at 6h, 24, 48h and 72h and the results are expressed as total flux (ph/s) in function of time (hours) post the injection of LNPs/ mRNA-Luc. A buffer Tris/sucrose was used as control.
• Figure 3 shows scheme (4) of synthesis of compound (VI).
• Figure 4 shows scheme (5) of synthesis of compound (VII).
• Figure 5 shows scheme (7) of synthesis of compound (VIII).
• Figure 6 shows scheme (8) of synthesis of compound (XV).
• Figure 7 shows scheme (9) of synthesis of compound (XVII).
• Figure 8 shows scheme (10) of synthesis of compound (XIX).
• Figure 9 shows scheme (11) of synthesis of compound (XXIII).
• Figure 10 shows scheme (12) of synthesis of compound (XXIX).
• Figure 11 shows scheme (13) of synthesis of compound (XXXI).
• Figure 12 shows scheme (14) illustrating the cleavage of lipidic compound of formula (III) (DOG-Cleave) through a cyclisation process generating successively “DOG-cleave transient” and the uncharged lipid “DOG-OH” in the final LNP formulation.
• Figure 13 shows a chromatogram illustrating the separation of the lipidic compound of formula (III) (DOG-Cleave), DOG-Cleave transient, DOG-OH, DSPC, Chol and DMG- PEG2000 on the C18-HPLC column.
• Figure 14 shows the HI responses induced by LNPs comprising influenza HA mRNA (1MpU-modified from Amptec) in non-human primates immunized twice four weeks apart (D0, D28) with 50 ⁇ g of mRNA in LNPs (LNPs (III)/DOG-CLEAVE or LNPs-L319)) injected IM.
• LNPs comprising influenza HA mRNA (1MpU-modified from Amptec) in non-human primates immunized twice four weeks apart (D0, D28) with 50 ⁇ g of mRNA in LNPs (LNPs (III)/DOG-CLEAVE or LNPs-L319)) injected IM.
• FIG. 15 Hemagglutination inhibiting antibody mean titers (HI titers) measured in sera collected at D21 in mice immunized at D0 and D21 with LNPs L319, LNPs (III) [DOG- Cleave], LNPs (XXI), and LNPs (XIX) containing DSPC as neutral lipid and loaded with mRNA encoding full-length hemagglutinin (HA) of influenza virus strain A/Netherlands/602/2009 (H1N1).
• HA hemagglutininin
• cleavable radical means that said radical, when covalently linked to another functional group for forming a compound, for example a lipidic compound as disclosed herein, is capable of being cleaved from the rest of the molecule, upon exposure to biological conditions, and such as in the context of the instant disclosure, upon exposure to a pH greater than 5 and, for example greater than 6.
• terminal radical means that said radical is a head-group or a tail-group.
• salts includes for example acid addition salts of compounds as disclosed herein derived from the combination of such compounds with non-toxic acid.
• acid addition salts include inorganic acids such as hydrochloric, hydrobromic, hydroiodic, sulfuric, nitric and phosphoric acid, as well as organic acids such as acetic, citric, propionic, tartaric, glutamic, salicylic, oxalic, methanesulfonic, para- toluenesulfonic, succinic, and benzoic acid, and related inorganic and organic acids.
• the pharmaceutically acceptable salts of compounds as disclosed herein can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, ethyl acetate and the like. Mixtures of such solvates can also be prepared.
• the source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent. Such solvates are within the scope of the present disclosure.
• - C t -C z a carbon chain that can have from t to z carbon atoms, where t and z may have the values from 1 to 7; for example, C 1 -C 4 is a carbon chain that may have from 1 to 4 carbon atoms; - a heteroatom is understood to mean nitrogen, oxygen or sulphur; - an heteroaromatic ring denotes a 5- or 6-membered aromatic ring comprising 1 or 2 heteroatoms; - an aromatic ring refers to a mono or polycyclic, such as a monocyclic, aromatic hydrocarbon radical of 6-20 atoms, for example of 6 atoms, derived by the removal of one hydrogen from a carbon atom of a parent aromatic ring system.
• An aromatic ring according to the disclosure is for example a phenyl group;
• the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.
• the term “about” or “approximately” as used herein refer to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. In some embodiments, the term “about” refers to ⁇ 10% of a given value.
• an antigen comprises any molecule, for example a peptide or protein, which comprises at least one epitope that will elicit an immune response and/or against which an immune response is directed.
• an antigen is a molecule which, optionally after processing, induces an immune response, which is for example specific for the antigen or cells expressing the antigen. After processing, an antigen may be presented by MHC molecules and reacts specifically with T lymphocytes (T cells).
• an antigen or fragments thereof should be recognizable by a T cell receptor and should be able to induce in the presence of appropriate co-stimulatory signals, clonal expansion of the T cell carrying the T cell receptor specifically recognizing the antigen or fragment, which results in an immune response against the antigen or cells expressing the antigen.
• any suitable antigen may be envisioned which is a candidate for an immune response.
• An antigen may correspond to or may be derived from a naturally occurring antigen. Such naturally occurring antigens may include or may be derived from allergens, viruses, bacteria, fungi, parasites and other infectious agents and pathogens or an antigen may also be a tumor antigen.
• aqueous solution or “aqueous solvent” refers to a composition comprising water.
• cationic refers to an ion or group of ions having a positive charge. It is understood that aspects and embodiments of the present disclosure described herein include “having,” “comprising,” “consisting of,” and “consisting essentially of” aspects and embodiments.
• the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of the stated element(s) (such as a composition of matter or a method step) but not the exclusion of any other elements.
• the term “consisting of” implies the inclusion of the stated element(s), to the exclusion of any additional elements.
• the term “consisting essentially of” implies the inclusion of the stated elements, and possibly other element(s) where the other element(s) do not materially affect the basic and novel characteristic(s) of the disclosure.
• the term “comprise” may also specify strictly the stated features, integers, steps or components, and therefore in such case it may be replaced with “consist”.
• the term “charged lipid” refers to any of a number of lipid species that exist in either a positively charged or negatively charged form within a useful physiological range e.g. pH ⁇ 3 to pH ⁇ 9. Charged lipids may be synthetic or naturally derived.
• Examples of charged lipids include phosphatidylserines, phosphatidic acids, phosphatidylglycerols, phosphatidylinositols, sterol hemisuccinates, dialkyl trimethylammonium-propanes, (e.g. DOTAP, DOTMA), dialkyldimethylaminopropanes, ethyl phosphocholines, dimethylaminoethane carbamoyl sterols (e.g. DC-Chol).
• DOTAP phosphatidylglycerols
• phosphatidylinositols sterol hemisuccinates
• dialkyl trimethylammonium-propanes e.g. DOTAP, DOTMA
• dialkyldimethylaminopropanes ethyl phosphocholines
• dimethylaminoethane carbamoyl sterols e.g. DC-Chol
• neutral lipid refers to any of a number of lipid species that is either not ionizable or is a neutral zwitterionic compound at a selected pH, for example at physiological pH.
• lipids include, but are not limited to, phosphatidylcholines, phosphatidylethanolamines sphingomyelins (SM), or neutral sphingolipids such as ceramides.
• Neutral lipids may be synthetic or naturally derived.
• the term “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.
• the term “lipid” refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are generally characterized by being poorly soluble in water, but soluble in many organic solvents.
• Lipid is a generic term encompassing fats, fatty oils, essential oils, waxes, phospholipids, glycolipids, sulfolipids, aminolipids, chromolipids (lipochromes), and fatty acids.
• lipid encompasses neutral lipids, steroid alcohol or ester thereof, and PEGylated lipids.
• lipid nanoparticle refers to particles having at least one dimension on the order of nanometers (e.g., 1-1000 nm) which may be formulated with at least one of the lipidic compound as disclosed herein.
• lipid nanoparticles are included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid to a target site of interest (e.g., cell, tissue, organ, tumor, and the like).
• lipid nanoparticles typically comprise a lipidic compound as disclosed herein, or one lipid derived from the hydrolysis of a lipidic compound as disclosed herein, and at least one ingredient selected from neutral lipids, steroid alcohols or esters thereof, and polymer conjugated lipids.
• lipid encapsulated refers to a lipid nanoparticle that provides an active agent or therapeutic agent, such as a nucleic acid with full encapsulation, partial encapsulation, or both.
• the polynucleotide is fully encapsulated in the lipid nanoparticle.
• head-group and tail-group describe parts of the compounds of the present disclosure, such as functional groups of such compounds. They are used to describe the orientation of one or more functional groups relative to other functional groups in said compounds. They are both “end group”.
• lipophilic or hydrophobic tail-group indicate in qualitative terms that the tail the tail has an affinity for lipids (and typically is lipid-soluble) and is water-avoiding (and typically is not water soluble).
• PEGylated lipid refers to a molecule comprising both a lipid portion and a polyethylene glycol portion.
• Pegylated lipids are known in the art and include 1 - (monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG) and the like.
• PEG-DMG polynucleotide
• oligonucleotides are used interchangeably. They refer to a polymeric form of at least two nucleotides, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Nucleic acids may have any three-dimensional structure, and may perform any function, known or unknown. They may be linear or cyclic.
• polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, closed-ended DNA (ceDNA), self-amplifying RNA (saRNA), stranded DNA (ssDNA), small interfering RNA (siRNA) and micro RNA (miRNA), recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
• loci locus defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, closed-ended DNA (ceDNA), self-amplifying RNA (saRNA), stranded DNA (s
• a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
• the sequence of nucleotides may be interrupted by non-nucleotide components.
• a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
• the term “complement of a polynucleotide” denotes a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence, such that it could hybridize with a reference sequence with complete fidelity.
• “Recombinant” as applied to a polynucleotide means that the polynucleotide is the product of various combinations of in vitro cloning, restriction and/or ligation steps, and other procedures that result in a construct that can potentially be expressed in a host cell.
• the term “steroid alcohol” or “sterol” refers to a group of lipids comprised of a sterane core bearing a hydroxyl moiety. As example of steroid alcohol, one may cite cholesterol, campesterol, sitosterol, stigmasterol and ergosterol. Esters of steroid alcohol or of sterol refer to ester of carboxylic acid with the hydroxyl group of the steroid alcohol.
• Suitable carboxylic acid comprises, further to the carboxyl moiety, a saturated or unsaturated, linear or branched, alkyl group.
• the alkyl group may be a C 1 -C 20 alkyl group.
• the carboxylic acid may be a fatty acid.
• the terms “prevent”, “preventing” or “delay progression of” (and grammatical variants thereof) with respect to a disease or disorder relate to prophylactic treatment of a disease, e.g., in an individual suspected to have the disease, or at risk for developing the disease.
• Prevention may include, but is not limited to, preventing or delaying onset or progression of the disease and/or maintaining at least one symptom of the disease at a desired or sub-pathological level.
• the term “prevent” does not require the 100% elimination of the possibility or likelihood of occurrence of the event. Rather, it denotes that the likelihood of the occurrence of the event has been reduced in the presence of a composition or method as described herein.
• the term “significantly” used with respect to change intends to mean that the observe change is noticeable and/or it has a statistic meaning.
• the term “substantially” used in conjunction with a feature of the disclosure intends to define a set of embodiments related to this feature which are largely but not wholly similar to this feature.
• target cells or “targeted cells” refer to cells of interest.
• the cells may be found in vitro, in vivo, in situ or in the tissue or organ of an organism.
• the organism may be an animal, such as a mammal, for example a human, and for example a human patient.
• the terms “treat” or “treatment” or “therapy” in the present text refers to the administration or consumption of a composition as disclosed herein with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect a disorder, the symptoms of the condition, or to prevent or delay the onset of the symptoms, complications, or otherwise arrest or inhibit further development of the disorder in a statistically significant manner.
• the terms “therapeutically effective amount” and “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes considered.
• the specific amount that is therapeutically effective can be readily determined by an ordinary medical practitioner and may vary depending on factors such as the type and stage of pathological processes considered, the patient’s medical history and age, and the administration of other therapeutic agents.
• the list of sources, ingredients, and components as described hereinafter are listed such that combinations and mixtures thereof are also contemplated and within the scope herein. It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein.
• Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein.
• Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
• All lists of items, such as, for example, lists of ingredients are intended to and should be interpreted as Markush groups. Thus, all lists can be read and interpreted as items “selected from the group consisting of’ ... list of items ... “and combinations and mixtures thereof.”
• Referenced herein may be trade names for components including various ingredients utilized in the present disclosure. The inventors herein do not intend to be limited by materials under any particular trade name.
• lipidic compounds of the disclosure are ionizable, and for example are cationic lipidic compounds.
• the lipidic compounds of the present disclosure may have asymmetric centers, chiral axes, and chiral planes (as described in: E. L. Eliel and S. H.
• cationic lipids disclosed herein may exist as tautomers and both tautomeric forms are intended to be encompassed by the scope of the disclosure, even though only one tautomeric structure is depicted.
• the pharmaceutically acceptable salts of lipidic compounds of the present disclosure have one or several counter ions which are generally physiologically acceptable.
• lipidic compounds as disclosed herein and the pharmaceutically acceptable salts thereof can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, ethyl acetate and the like. Mixtures of such solvates can also be prepared.
• solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.
• solvates are within the scope of the present disclosure.
• the lipidic compounds as disclosed herein have one hydrophilic head group formed by one radical of formula (I) also named terminal radical to illustrate the fact that it is linked, directly or not, to an end of one hydrophobic or lipophilic tail also forming said lipidic compounds.
• the radical of formula (I) has the following definition: Y-(CHR) n -Z-(CHR’) p -Q * (I) wherein: - * is the end linked, directly or not, to one C 10 to C 60 and preferably to C 10 to C 55 lipophilic or hydrophobic tail-group, - Y is a radical selected in the group consisting of a C 1 -C 5 alkyl, a C 1 -C 5 alkoxy, a C 1 -C 5 acyl, a C 1 -C 5 hydroxyalkyl, a C 1 -C 5 aminoalkyl, a C 1 -C 5 alkylcarboxyl ester, an acetamido, an N,N- C 1 -C 5 alkylamido, a C 1 -C 5 fluoroalkyl, for example a C 1 -C 5 perfluoroalkyl, for example a trifluoromethyl, an imidazolyl, a
• the radical of formula (I) exists under a protonated and stabilized form at a pH lower than 6.0 and for example lower than 5.5 and for example lower than 5.
• a radical of formula (I) present in a lipidic compound according to the disclosure, advantageously undergoes a chemical rearrangement. As shown in the following scheme 1, this chemical rearrangement leads to its cleavage from the rest of the lipidic molecule.
• This ability to exist in a positively charged form is for example efficient for immobilizing negatively charged nucleic acids and charging them in specific chemical vehicles dedicated to promoting the, in vitro or in vivo, targeted release of said nucleic acids.
• Z and Q are both one radical -NH-CH 2 -CO-O-, and for example n and p are both 2.
• Z and Q are different and one of them is one radical -NH-CH 2 -CO-O- and the other one is one radical -CH(NH 2 )-CO-O-, and for example n and p are different and equal to 1 or 2.
• Z is preferably -CR’’(NH 2 )- CO-O- and Q is -NH-CH 2 -CO-O-.
• Z and Q are both one radical -CR’’(NH 2 )- CO-O- with preferably R’’ being an hydrogen and for example n and p are both 1.
• one radical of formula (I) is directly or not, attached to a hydrophobic (lipophilic) tail group (e.g, a covalent bond).
• the hydrophobic or lipophilic tail is generally in C 10 to C 55 but may be also in C 10 to C 60 .
• hydrocarbon skeleton that is optionally interrupted by one or several atoms of oxygen or nitrogen and/or one or several -O-CO- or -CO-O- groups and which one nitrogen atom present on the skeleton can be, linked, directly or not, to the radical represented by formula (I).
• the hydrophobic or lipophilic tail is an optionally substituted branched or unbranched linear saturated or unsaturated C 10 to C 60 and preferably C 10 to C 55 hydrocarbon radical, and which hydrocarbon skeleton that is optionally interrupted by one or several atoms of oxygen and/or one or several -O-CO- or -CO-O- groups and if one nitrogen atom is present in the skeleton it is present under a form that cannot be protonated and is linked, directly or not, and preferably directly linked to the spacer, A.
• the hydrophobic or lipophilic tail comprises at least two, three or more hydrocarbon chains each one independently being selected from optionally substituted C 8 -C 24 , for example C 10 -C 20, alkyl chain, optionally substituted variably saturated or unsaturated C 8 -C 24, for example C 10 -C 20, alkenyl chain and optionally substituted, saturated, variably saturated or unsaturated C 8 -C 24 , for example C 10 -C 20 , acyl chain with said alkyl, alkenyl or acyl chains can be interrupted by one or several atoms of oxygen and/or one or several moieties like -O-CO- or -CO-O-.
• Each hydrocarbon chain may be substituted by at least one radical selected from -OH, CO 2 H and alkyl group in C 1 to C 4 and preferably being unsubstituted.
• the hydrophobic or lipophilic tail is selected in the group consisting of
• the hydrophobic or lipophilic tail comprises at least three, four or more hydrocarbon chains, each one independently being selected from optionally substituted C 4 -C 24 , for example C 5 -C 20 , alkyl chain and optionally substituted C 4 -C 24 , for example C 10 -C 20 , alkenyl chain.
• the hydrophobic or lipophilic tail comprises at least two, three, four or more hydrocarbon C 4 -C 24 chains, with at least one chain and preferably at least two chains being interrupted by at least one oxygen atom and/or at least one moiety selected among —O—(O ⁇ C)— and —(C ⁇ O)—O—.
• the hydrophobic or lipophilic tail comprises at least three, four or more hydrocarbon chains, with at least two chains being optionally substituted C 4 -C 24 , for example C 5 -C 20 alkylene chain with optionally each one being or not independently interrupted by at least one moiety selected among —O—(O ⁇ C)— and —(C ⁇ O)—O—.
• the hydrophobic or lipophilic tail comprises at least three, four or more hydrocarbon chains, with all chains being optionally substituted C 4 -C 24 , for example C 5 -C 20 alkyl chains with optionally each one being or not independently interrupted by at least one moiety selected among —O—(O ⁇ C)— and —(C ⁇ O)—O—.
• the hydrophobic or lipophilic tail is the tail (R1a) or (R1b) also respectively named DOG alkyl or DOG ether.
• the cationic and/or ionizable lipidic compound of the disclosure is a compound of formula (II) Y-(CHR) n -Z-(CHR’) p -Q-A-R 1 (II) wherein: - Y, R, n, Z, R’, p and Q are as defined in claim 1; - R 1 is a C 10 to C 60 and preferably a C 10 to C 55 lipophilic or hydrophobic tail-group in particular as described herein; and A is a spacer arm having from 2 to 24, for example from 2 to 18, for example from 4 to 12 carbon atoms, or for example from 2 to 12 carbon atoms, in a branched or unbranched linear saturated or unsaturated hydrocarbon chain, said chain being interrupted by one or several atoms of oxygen and/or moieties selected among —S—S— ; —(C ⁇ O)—O—;—O—(O ⁇ C)—; —(C ⁇ O)—NH
• Z and Q are both one radical -NH-CH 2 -CO-O-.
• Z and Q are different and one of them is one radical -NH-CH 2 -CO-O- and the other one is one radical -CH(NH 2 )-CO-O- and preferably Z is -CR’’(NH 2 )-CO-O- with R’’ being preferably a hydrogen atom, and Q is -NH-CH 2 -CO-O-.
• Z and Q are both one radical -CR’’(NH 2 )- CO-O-.
• the lipidic compound as disclosed herein is a compound of formula (IIa) Y-(CHR) n -NH-CH 2 -CO-O-(CHR’) p -NH-CH 2 -CO-O-A-R 1 (IIa) wherein: - Y, R, n, R’, R 1 , A and p are as previously defined in formula (I) or (II); or one of its pharmaceutically acceptable salts and any of its racemic, enantiomeric and diastereoisomeric isomer forms.
• n and p are both 2.
• Y is a radical methoxy.
• the spacer arm A of formula (II) and (IIa) it is like the ones conventionally considered in the field of the lipidic compounds. Accordingly, the choice of such spacer arm does not raise any difficulty for the man skilled in the art. Naturally, it needs to be inert or not prejudicial to the efficiency of the lipidic compound.
• the spacer arm A has 2 to 24 and for example from 2 to 12 carbon atoms, or for example from 4 to 10 carbon atoms, comprises at least one or several ethylene oxide units and optionally one or several moieties selected among -OCO-; COO-, -NHCOO-, -OCONH- and -SS-.
• spacer arms convenient for the disclosure, it may be cited the following ones and which right end being the one to be linked to the lipophilic or hydrophobic tail-group: O O O O O O O O O O O O O
• the spacer consists in ethylene oxide units.
• the spacer is a poly(ethylene oxide) (aka polyethylene glycol - PEG).
• the spacer may comprise 1 to 24 ethylene oxide units and for example 2, 3, 4, 5, 6, 7, 8, 10, 12 and 24 ethylene oxide units.
• the spacer comprises a poly(ethylene oxide) moiety and further includes at least one moiety selected among -COO- ,-OCO-, -NHCOO-, -OCONH- and -CH 2 CH 2 -.
• the compound is of formula (II) wherein Z is one radical -CH(NH 2 )-CO-O-.
• the compound is one radical -NH-CH 2 -CO-O- and in particular is the following compound (VI): (VI) or one of its salts, for example its trifluoroacetate salt, and or one of its racemic, enantiomeric and diastereoisomeric isomer forms.
• the compound is of formula (II) wherein Z and Q are both one radical -NH-CH 2 -CO-O-.
• the compound of formula (II) may be selected among the following compounds (III) and (VI) to (XXXII), and for example among compounds (III), (VI), (XVII), (XIX), (XXI), (XXII), (XXIV), (XXVII) and (XXVIII), or for example among compounds (III), (XIX) or (XXI), and for example may be compound of formula (III) (also named DOG-CLEAVE) and their salts, for example their trifluoroacetate salt, and or their racemic, enantiomeric and diastereoisomeric isomer forms:
• a secondary amino moiety may be indifferently written -NH- or -N-.
• the compounds (III), (XVII), (XIX), (XXI), (XXII), (XXIV), (XXVII) and (XXVIII), are for example efficient for the formulation of stable LNPs capable of delivering a functional mRNA into target tissues after parenteral administrations and for the induction of the expression of a protein such as EPO or of an immune response in the case the delivered mRNA codes for an antigen.
• Preparation of lipidic compounds The lipidic compounds according to the disclosure can be prepared from readily commercially available or described in the literature starting materials using methods and procedures known from the skilled person.
• these lipidic compounds may be obtained by covalent coupling, between a precursor of the radical of formula (I) and a lipidic compound or derivative thereof having a terminal reactive group able to react with said precursor.
• This terminal reactive group may be located directly on the end of the hydrophobic or lipophilic part of the lipidic compound to transform or on the end of a spacer arm already linked to the hydrophobic or lipophilic part of the lipid compound.
• the choice of the convenient precursor of the radical of formula (I) intended to react with the lipidic compound for forming the expected covalent linkage is clearly within the competence of the man skilled in the art.
• the precursor only needs to have a group able to chemically react with the one of the lipidic compound for forming the covalent linking.
• radical of formula (I) precursor of the radical of formula (I) and the lipidic compound or derivative thereof to transform they may be easily produced by a man skilled in the art, for example according to the methods of preparation submitted in the following examples.
• a convenient precursor for a radical of formula (I) it can be cited the [2-[2(2-methoxyethyl )amino]acetyl]oxyethyl]amino]acetic acid or also named 2-[tert- butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl] amino]acetic acid, and for example its protected form of formula
• Scheme 1 One specific approach for preparing such a precursor of the radical of formula (I) is depicted in Scheme 1 below.
• the covalent coupling between such a precursor may be further performed according to methods that are known to those skilled in the art in regard of the chemical nature of the reactive group of the precursor of the radical of formula (I) and the one on the lipidic compound or derivative thereof to be transformed.
• the covalent linking may be formed for example by esterification, amidation, or carbamation.
• Scheme 2 One specific approach for this covalent coupling is depicted in Scheme 2 below for the synthesis of the trifluorocetate salt of the lipidic compound (III) wherein the coupling is obtained with an esterification reaction.
• Lipid nanoparticles (LNPs) manufacturing methods The present disclosure relates to methods for manufacturing lipid nanoparticles using the lipidic compounds as disclosed herein.
• the disclosure relates to a method for manufacturing lipid nanoparticles containing a nucleic acid, wherein the method comprises at least the steps of: a) solubilizing, in a water miscible organic solvent, at least one lipidic compound as disclosed herein, b) mixing the organic solvent obtained at step a) with an aqueous solvent buffered at a pH ranging from about 3.0 to about 4.5 and comprising at least one nucleic acid, and c) obtaining said lipid nanoparticles containing the nucleic acid in the aqueous solvent.
• a method for manufacturing lipid nanoparticles as disclosed herein may comprise at least steps of: a) solubilizing, in a water miscible organic solvent, at least one lipidic compound as disclosed herein and at least one lipid selected from the group consisting of neutral lipids, steroid alcohols or esters thereof, and PEGylated lipids, b) mixing the organic solvent obtained at step a) with an aqueous solvent comprising at least one nucleic acid, and c) obtaining lipid nanoparticles containing the nucleic acid in the aqueous solvent.
• the lipidic compound of the disclosure may be present in an amount sufficient to structure the lipid nanoparticles and to encapsulate any loads to be encapsulated.
• the amount of ionizable lipidic compound to be used in the lipid nanoparticles may be determined by the skilled person according to any known techniques and is adapted according to the nature and amount of the load, and nature and amount of other lipids susceptible to be present.
• the step a) further comprises solubilizing in the organic solvent at least one lipid selected from the group consisting of neutral lipids, steroid alcohols or esters thereof, and PEGylated lipids.
• Neutral lipids, steroid alcohols or esters thereof, and PEGylated lipids suitable for the disclosure may be as described below.
• the step a) may further comprise solubilizing in the organic solvent at least one neutral lipid, at least one steroid alcohol or an ester thereof, and at least one PEGylated lipid, and wherein said lipidic compound, said neutral lipid, said steroid alcohol or an ester thereof, and said PEGylated lipid are present in the organic solvent at a molar amount of about 30% to about 70% of lipidic compound, of about 0% to about 50% of neutral lipid, of 20% to about 50% of steroid alcohol or an ester thereof, and of about 1% to about 15% of PEGylated, relative to the total amount of lipid and lipidic compound.
• Useful water-miscible organic solvents may be any water-miscible organic solvent capable to solubilize the lipidic compound as disclosed herein and any other added lipids.
• suitable organic solvents one may cite ethanol or methanol, 1-propanol, isopropanol, t-butanol, THF, DMSO, acetone, acetonitrile, diglyme, DMF, 1-4 dioxane, ethylene glycol, glycerine, hexamethylphosphoramide, hexamethylphosphorous triamide.
• the organic solvent may be ethanol and isopropanol.
• Aqueous solvents usable at step b) include aqueous buffered solutions.
• aqueous buffered solution examples include acidic buffer, such as include citrate buffer, sodium acetate buffer, succinate buffer, borate buffer or a phosphate buffer.
• an aqueous buffered solvent may be a citrate buffered solution or an acetate buffered solution.
• the pH of the aqueous solvent may range from about 3.0 to about 4.5, for example from about 3.5 to about 4.5, and for example at about 4.0.
• the organic and aqueous solvents may be mixed at a ratio organic solvent:aqueous solvent ranging from about 1:1 to about 1:6. In one embodiment, the ratio may range from about 1:2 to about 1:4, and for example may be a ratio of about 1:3.
• the water miscible organic solvent and the aqueous solvent may be mixed at step b) at a flow rate ranging from about 0.01 ml/min to about 12 ml/min.
• the flow rate may range from about 0.02 ml/min to about 10 ml/min, from about 0.5 ml/min to about 8 ml/min, from about 1 ml/min to about 6 ml/min, or at about 4 ml/min.
• the step of mixing may be carried by any known method in the art. For instance, both solvents may be mixed with a T-tube or a Y-connector.
• the mixing may be carried out by laminar flow mixing with a microfluidic micromixer as described by Belliveau et al. (2012).
• the aqueous solvent at step b) comprises a nucleic acid.
• a nucleic acid may encode at least one antigen.
• a suitable nucleic acid may be for example as detailed herein.
• the method may further comprise a step of increasing the pH from acidic to neutral or slightly above neutral.
• the method may comprise a step d) of increasing the pH of the aqueous solvent containing the lipid nanoparticles obtained at step c) at a pH ranging from about 5.0 to about 8.5, for example from about 5.5 to about 8.0, for example from about 6.0 to about 7.5, and for example from about 6.5 to about 7.0.
• the step of increasing the pH may be carried by any known method in the art.
• the change in pH may carried by a dialyzing or diafiltration step.
• step d) of the method of the disclosure may further comprise at least one step of dialyzing or diafiltrating the lipid nanoparticles.
• the dialysis or diafiltration step may be made against an aqueous solvent with a pH ranging from about 5.0 to about 8.5, for example from about 5.5 to about 8.0, for example from about 6.0 to about 7.5, and for example from about 6.5 to about 7.0.
• a pH ranging from about 5.0 to about 8.5, for example from about 5.5 to about 8.0, for example from about 6.0 to about 7.5, and for example from about 6.5 to about 7.0.
• the increase of the pH from acidic (i.e., from about 3.0 to about 4.5) to more neutral, or slightly above neutral, pH (i.e., from about 5.0 to about 8.5) advantageously results into the cleavage and self-rearrangement of the ionizable lipidic compounds as disclosed herein into lipidic compounds of formula (IV), (Va) or (Vb) as detailed below.
• An aqueous solvent usable at step d) may further contain a carbohydrate to improve stabilization of the lipid nanoparticles and osmolarity of the solution.
• Suitable carbohydrate may be sucrose, mannitol, glucose, dextrose or trehalose.
• the carbohydrate may be present in an amount, relative to the total amount of the aqueous solvent, of about 5% to about 10%, and for example at about 8%.
• step d) of the method as disclosed herein may comprise at least two steps of dialyzing the lipid nanoparticles.
• a first dialyzing step may be made against a similar aqueous solvent (similar in terms of pH and content) and may remove the organic solvent.
• a second dialysis step may be made against a different aqueous solvent (different in term of pH and possibly in term of content).
• a pH of the dialyzing solution may range from about 5.5 to about 7.5, for example from about 6.0 to about 7.0, for example from about 6.5 to about 7.0, and for example at about 6.5.
• the dialyzing solution of the second dialysis may be a buffer solution, for example a phosphate buffer, a TRIS buffer, a Hepes buffer, a histidine buffer, or a glycine buffer.
• Osmolarity of the buffer may be adjusted with a salt, such as NaCl, or with a carbohydrate, such as glycerol, sucrose, mannitol, glucose, dextrose or trehalose. In one embodiment, osmolarity is adjusted to reach a final osmolality close to 290 mOsmol/kg as to inject isotonic solution into the body.
• a method may comprise any further step suitable to harvest, purify, concentrate and/or sterilize the lipid nanoparticles to further formulate them as a pharmaceutical composition, for example as an immunogenic composition.
• the disclosure relates to lipid nanoparticles obtainable according to a manufacturing method as disclosed herein.
• the disclosure relates to a method for manufacturing a pharmaceutical composition comprising at least the steps of: i) manufacturing at least one lipid nanoparticle according to the method as disclosed herein, and ii) combining the lipid nanoparticles obtained at step i) with at least one pharmaceutically acceptable excipient or carrier.
• the disclosure relates to a method for manufacturing an immunogenic composition
• a method for manufacturing an immunogenic composition comprising at least the steps of: i) manufacturing at least one lipid nanoparticle according to the method as disclosed herein, said lipid nanoparticle containing at least one nucleic acid encoding for at least one antigen, and ii) combining the lipid nanoparticles obtained at step i) with at least one pharmaceutically acceptable excipient or carrier.
• the pharmaceutical and immunogenic compositions suitable for the disclosure are more detailed thereafter.
• the lipid nanoparticles of the disclosure may be manufactured with a lipidic compound of formula (I), (II) or (IIa), and for example of formula (III) to (XXXII), for example of formula (III), (VI), (XVII), (XIX), (XXI), (XXII), (XXIV), (XXVII) and (XXVIII), and for example of formula (III), (XIX) or (XXI), and for example with a lipidic compound of formula (III).
• the lipid nanoparticles as disclosed herein may be manufactured with a neutral lipid that is DSPC or DOPE, a steroid alcohol that is cholesterol, and a PEGylated lipid that is PEG-PE (PEG2000-PE) or DMG-PEG (DMG- PEG2000).
• the lipid nanoparticles as disclosed herein may be manufactured with a lipidic compound of formula (III) to (XXXII), a neutral lipid that is DSPC, a steroid alcohol that is cholesterol, and a PEGylated lipid that is PEG-PE (PEG2000-PE).
• the lipid nanoparticles as disclosed herein may be manufactured with a lipidic compound of formula (III), (VI), (XVII), (XIX), (XXI), (XXII), (XXIV), (XXVII) and (XXVIII), a neutral lipid that is DSPC, a steroid alcohol that is cholesterol, and a PEGylated lipid that is PEG-PE (PEG2000-PE).
• a lipidic compound of formula (III), (VI), (XVII), (XIX), (XXI), (XXII), (XXIV), (XXVII) and (XXVIII) a neutral lipid that is DSPC, a steroid alcohol that is cholesterol, and a PEGylated lipid that is PEG-PE (PEG2000-PE).
• the lipid nanoparticles as disclosed herein may be manufactured with a lipidic compound of formula (III), (XIX) or (XXI), a neutral lipid that is DSPC, a steroid alcohol that is cholesterol, and a PEGylated lipid that is PEG-PE (PEG2000- PE).
• the lipid nanoparticles as disclosed herein may be manufactured with a lipidic compound of formula (III), a neutral lipid that is DSPC, a steroid alcohol that is cholesterol, and a PEGylated lipid that is PEG-PE (PEG2000-PE).
• Lipid nanoparticles Lipid nanoparticles (LNPs)
• the disclosure relates to lipid nanoparticles comprising at least lipidic compound of formula (IV): HO-L (IV) wherein L is a lipophilic or hydrophobic tail-group as described herein, for example a C 10 to C 55 lipophilic or hydrophobic tail-group, and at least one nucleic acid.
• the lipid nanoparticles as disclosed herein may comprise at least one lipidic compound of formula (Va) or (Vb): HO-R 1 (Va) or OH-A-R 1 (Vb) wherein R1and A are as defined previously.
• the lipid nanoparticles as disclosed herein may comprise at least one lipid selected from the group consisting of neutral phospholipids or sphingolipids, steroid alcohols or esters thereof, and PEGylated lipids.
• the lipidic compounds formula (IV), (Va) or (Vb) result from the cleavage and self- rearrangement of the ionizable lipidic compound as disclosed herein when the lipid nanoparticles are brought from acidic pH (i.e. from about 3.0 to about 4.5) to more neutral or slightly above neutral pH (i.e. from about 5.0 to about 8.5). Those lipidic compounds are neutral, non-ionizable compounds.
• the lipid nanoparticles may have a diameter making them suitable for systemic, for example parenteral, or for intramuscular, intradermic, or subcutaneous administration.
• the lipid nanoparticles have a Z-average size of less than 600 nanometers (nm), for example of less than 400 nm.
• the LNPs have a Z-average size of less than 200 nm.
• Such size is advantageously compatible with sterile filtration and most appropriate for migration through the lymphatic vessels after intramuscular or subcutaneous administration. This size is also appropriate for intravenous administration, since larger particle injection could induce capillary thrombosis.
• the lipid nanoparticles may have a Z-average diameter size in the range of from about 20 nm to about 300 nm, for example from about 20 nm to about 250 nm, for example about 30 nm to about 200 nm, about 40 nm to about 180 nm, from about 60 nm to about 170 nm, from about 80 to about 160 nm, and from about 90 to about 150 nm.
• the nanoparticles may have a diameter in the range of about 90 to about 150 nm.
• the “Z-average size” of the lipid nanoparticles may be determined by dynamic light scattering (DLS).
• the Z-Average size or Z-Average mean used in dynamic light scattering is a parameter also known as the cumulants mean. It is the primary and most stable parameter produced by the technique.
• the Z-Average mean is defined as the ‘harmonic intensity averaged particle diameter’.
• size may be determined by filtration screening assays.
• a particle preparation is less than a stated size, if at least 90%, for example at least 95%, and for example at least 97% of the particles pass through a “screen-type” filter of the stated size.
• the “polydispersity index” (PI) is a measurement of the homogeneous or heterogeneous size distribution of the individual lipid nanoparticles in a lipid nanoparticles mixture and indicates the breadth of the particle distribution in a mixture.
• the PI can be determined, for example, as described herein.
• the polydispersity index of the nanoparticles described herein as measured by dynamic light scattering is 0.5 or less, for example 0.4 or less, for example 0.3 or less, or even for example 0.2 or less.
• the lipid nanoparticles are colloidally stable in the sense that no, or substantially no, aggregation, precipitation or increase of size and polydispersity index as measured by dynamic light scattering may be observed over a given period of time, e.g. over at least two hours to over several months, for example at least 1, 2, 3, 4, 5, 6 or 12 months.
• the lipid nanoparticles may comprise or encapsulate at least one nucleic acid.
• the nucleic acid may be encapsulated in and/or adsorbed on an exterior surface of the lipid nanoparticles.
• the lipidic compound of formula (IV), (Va) or (Vb) may form a complex with and/or encapsulates the nucleic acid.
• the lipidic compound may be comprised in a vesicle encapsulating the nucleic acid.
• the lipid nanoparticles have a global surface charge which is the sum of the negative and positive electric charges at the surface of the particles, and which is represented by the zeta potential.
• the zeta potential is the potential difference between the dispersion medium and the stationary layer of fluid attached to the dispersed particle.
• Zeta potential is widely used for quantification of the magnitude of the electrical charge at the double layer.
• Zeta potential can be calculated using theoretical models and experimentally determined using electrophoretic mobility or dynamic electrophoretic mobility measurements. Electrophoresis may be used for estimating zeta potential of particulates.
• the zeta potential of a dispersion can be measured by applying an electric field across the dispersion. Particles within the dispersion with a zeta potential will migrate toward the electrode of opposite charge with a velocity proportional to the magnitude of the zeta potential. This velocity may be measured using the technique of the Laser Doppler Anemometer.
• the frequency shift or phase shift of an incident laser beam caused by these moving particles may be measured as the particle mobility, and this mobility may be converted to the zeta potential by inputting the dispersant viscosity and dielectric permittivity, and the application of the Smoluchowski theories.
• Electrophoretic velocity is proportional to electrophoretic mobility, which is the measurable parameter.
• Suitable systems such as the Nicomp 380 ZLS system or the Malvern nanoZS can be used for determining the zeta potential.
• Such systems usually measure the electrophoretic mobility and stability of charged particles in liquid suspension. These values are a predictor of the repulsive forces being exerted by the particles in suspension and are directly related to the stability of the colloidal system.
• the zeta potential of the lipid nanoparticles as disclosed herein is close to neutral. Indeed, at pH neutral, or slightly above neutral, (from 5.0/5.5 to 8.5), the ionizable, cleavable lipidic as disclosed herein has undergone a self-rearrangement resulting into the leave of the radical of formula (I) and to the remaining of the neutral, non-charged, hydrophobic or lipophilic tail group.
• to have a zeta potential close to zero facilitates particle mobility in the body, reduces opsonization and augment access to target tissues.
• the zeta potential of the lipid nanoparticles may be from about -3 mV to about +3 mV, for example from about -1 mV to about +1 mV, and for example from about -0.5 mV to about +0.5 mV.
• the lipid nanoparticles described herein can be formed by adjusting, at the time of the preparation, a positive to negative charge, depending on the charge ratio of the ionizable lipidic compound as disclosed herein (cationic charges from the quaternary ammonium: N of the terminal radical of formula (I)) to the nucleic acid (anionic charges from the phosphate : P) and mixing the nucleic acid and the lipidic compound.
• the charges of the ionizable lipidic compound and of the nucleic acid are charges at a selected pH, such as a pH of the formulating process, which is from about 3.0 to about 4.5.
• the nucleic acid amount and the lipidic compound amount can be easily determined by one skilled in the art in view of a loading amount upon preparation of the nanoparticles.
• the calculated charge ratio of positive charges to negative charges may range from about 1:1 to about 14:1, for example from about 2:1 to about 12:1, for example from about 4:1 to about 10:1, and for example from about 6:1 to about 8:1, and for example is about 6:1.
• lipid nanoparticles as disclosed herein encapsulating a nucleic acid may have a Z-average size of about 80-180 nm and a calculated charge ratio N/P of about 6- 12:1, for example of about 3-9:1.
• the lipid nanoparticles as disclosed herein may comprise at least one cleavable lipidic compound as disclosed herein.
• the lipid nanoparticles obtained according to the method as disclosed herein to a neutral pH, or slightly above neutral pH, (5.0/6.0 to 8.5) to cleave the ionizable cleavable cationic lipidic compound as disclosed herein, not all those compounds may be cleaved.
• the lipidic compound located within the lipid nanoparticles, that in the core of the LNPs may be protected from the change of pH and may not undergo to the cleaving process.
• the presence of the remaining cleavable cationic and/or ionizable lipidic compound as disclosed herein may be observed by known method in the art, such as TLC or HPLC analysis.
• the lipid nanoparticles as disclosed herein may further comprise at least one lipid selected from the group consisting of neutral lipids, steroid alcohols or ester thereof, and PEGylated lipids.
• Neutral lipids A composition or lipid nanoparticles as disclosed herein may include a neutral lipid. The presence of neutral lipids may improve structural stability of the lipid nanoparticles. The neutral lipid can be appropriately selected in view of the delivery efficiency of nucleic acid.
• the neutral lipids are distinct from the lipidic compound of formula (IV), (Va) or (Vb). Neutral lipids are either not ionizable or are neutral zwitterionic compounds at a selected pH.
• Neutral lipids useful for the disclosure may be selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, sphingomyelins, and ceramides.
• Phosphatidylcholines and phosphatidylethanolamines are zwitterionic lipids.
• Sphingomyelins and ceramides are not ionizable lipids.
• DSPC l,2-distearoyl-sn-glycero-3-phosphocholine
• DPPC l,2-dipalmitoyl-sn-glycero-3- phosphocholine
• DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine
• POPC 1,2-palmitoyl- 2-oleoyl-sn-glycero-3-phosphocholine
• DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine).
• phosphatidylethanolamines useful for the disclosure one may mention DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DPPE (l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DSPE (l,2-distearoyl-s/i-glycero-3-phosphoethanolamine), DLPE (l,2-dilauroyl-SM-glycero-3- phosphoethanolamine), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, or l-stearoyl- 2-oleoyl-phosphatidyethanolamine (SOPE).
• DOPE 1,2-dioleyl-sn-glycero-3-phosphoethanolamine
• DPPE l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine
• a neutral lipid may be selected from the group consisting of phosphatidylcholines, such as DSPC, DPPC, DMPC, POPC, DOPC; phosphatidylethanolamines, such as DOPE, DPPE, DMPE, DSPE, DLPE; sphingomyelins; and ceramides.
• a neutral lipid suitable for the disclosure may be DSPC, DOPC, and DOPE, and for example may be DSPC or DOPE.
• Neutral lipids may be present at step a) of the method for formulating the lipid nanoparticles as disclosed herein in a molar amount ranging from about 0% to about 50%, for example from about 5% to about 45%, for example from about 8% to about 40%, and for example from about 10% to about 30% relative to the total molar amount of the lipid and lipidic compound as disclosed herein.
• Neutral lipids may be present in the lipid nanoparticles as disclosed herein in a molar amount ranging from about 0% to about 50%, for example from about 5% to about 45%, for example from about 8% to about 40%, and for example from about 10% to about 30% relative to the total molar amount of the lipid and lipidic compound of formula (IV), (Va) or (Vb) which may be present in the lipid nanoparticles.
• Neutral lipids may be present in lipid nanoparticles as disclosed herein in a molar ratio lipidic compound of formula (IV), (Va) or (Vb):neutral lipid which may range from about 70:1 to about 1:2, for example from about 30:1 to about 1:1, for example from about 15:1 to about 2:1, for example from about 10:1 to about 4:1, and for example is about 5:1.
• Steroid alcohols or esters thereof A composition or lipid nanoparticles as disclosed herein may include a steroid alcohol (or sterol) or an ester thereof. The presence of sterol or an ester of sterol may improve structural stability of the lipid nanoparticles.
• Sterols or steroid alcohols useful for the disclosure may be selected from the group consisting of cholesterol or its derivatives, ergosterol, desmosterol (3ß-hydroxy-5,24- cholestadiene), stigmasterol (stigmasta-5,22-dien-3-ol), lanosterol (8,24-lanostadien-3b-ol), 7- dehydrocholesterol ( ⁇ 5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), zymosterol (5 ⁇ -cholesta-8,24-dien-3ß-ol), lathosterol (5 ⁇ -cholest-7-en-3ß-ol), diosgenin ((3 ⁇ ,25R)-spirost- 5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitostanol, campesterol (campest-5-en-3ß-ol), campestanol (5a-campestan-3b-ol), 24-m
• Esters of steroid alcohol or of sterol refer to ester of carboxylic acid with the hydroxyl group of the steroid alcohol.
• Suitable carboxylic acid comprises, further to the carboxyl moiety, a saturated or unsaturated, linear or branched, alkyl group.
• the alkyl group may be a C 1 -C 20 saturated or unsaturated, linear or branched, alkyl group, for example a C 2 -C 18 , for example a C 4 -C 16 , for example C 8 -C 12 saturated or unsaturated, linear or branched, alkyl group,
• the carboxylic acid may be a fatty acid.
• a fatty acid may be caprylic acid, capric acid, lauric acid, stearic acid, margaric acid, oleic acid, linoleic acid, or arachidic acid.
• an ester of sterol suitable for the disclosure may be a cholesteryl ester.
• Esters of sterol or of steroid alcohol useful for the disclosure may be selected from the group consisting of cholesteryl margarate (cholest-5-en-3ß-yl heptadecanoate), cholesteryl oleate, and cholesteryl stearate.
• Sterols or steroid alcohols or esters thereof useful for the disclosure may be selected from the group consisting of cholesterol or its derivatives, ergosterol, desmosterol (3ß-hydroxy- 5,24-cholestadiene), stigmasterol (stigmasta-5,22-dien-3-ol), lanosterol (8,24-lanostadien-3b- ol), 7-dehydrocholesterol, dihydrolanosterol (24,25-dihydrolanosterol), zymosterol (5 ⁇ - cholesta-8,24-dien-3ß-ol), lathosterol (5 ⁇ -cholest-7-en-3ß-ol), diosgenin ((3 ⁇ ,25R)-spirost-5- en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitostanol, campesterol (campest-5-en-3ß-ol), campestanol (5a-campestan-3b-ol), 24-methylene cholesterol
• a sterol useful for the disclosure may be a cholesterol derivative such as an oxidized cholesterol.
• Oxidized cholesterols suitable for the disclosure may be 25-hydroxycholesterol, 27- hydroxycholesterol, 20 ⁇ -hydroxycholesterol, 6-keto-5 ⁇ -hydroxycholesterol, 7-keto- cholesterol, 7 ⁇ ,25-hydroxycholesterol and 7 ⁇ -hydroxycholesterol.
• oxidized cholesterols may be 25-hydroxycholesterol and 20 ⁇ -hydroxycholesterol, and for example it may be 20 ⁇ -hydroxycholesterol.
• a sterol or steroid alcohol, or ester thereof, suitable for the disclosure may be cholesterol, a cholesteryl ester, or a cholesterol derivative, for example an oxidized cholesterol.
• a sterol or steroid alcohol, or ester thereof, suitable for the disclosure may be cholesterol or a cholesteryl ester, and for example may be cholesterol.
• Sterols or steroid alcohols, or esters thereof may be present at step a) of the method for formulating the lipid nanoparticles as disclosed herein in molar amount ranging from about 0 to about 60%, for example from about 10% to about 50%, and for example from about 20% to about 50% relative to the total molar amount of the lipid and ionizable lipidic compound as disclosed herein.
• Sterols or steroid alcohols, or esters thereof may be present in the lipid nanoparticles as disclosed herein in molar amount ranging from about 0 to about 60%, for example from about 10% to about 50%, and for example from about 20% to about 50% relative to the total molar amount of the lipid and lipidic compound of formula (IV), (Va) or (Vb) which may be present in the lipid nanoparticles.
• Sterols or steroid alcohols, or esters thereof may be present in lipid nanoparticles as disclosed herein in a molar ratio lipidic compound of formula (IV), (Va) or (Vb):steroid alcohol, or ester thereof, which may range from about 4:1 to about 1:2, for example from about 3.5:1 to about 1:1.8, for example from about 2:1 to about 1:1.5, for example from about 1.5:1 to about 1:1.2, and for example is about 1.3:1 to about 1:1.3.
• PEGylated lipids A composition or lipid nanoparticles as disclosed herein may include a PEGylated (or PEG-) lipid.
• Contemplated PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C 6 -C 20 length.
• the addition of PEG-modified lipids to a composition of lipid nanoparticles as disclosed herein may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the composition or lipid nanoparticles to the target cells.
• a suitable PEGylated lipid may be, for example, a pegylated diacylglycerol (PEG- DAG), such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (DMG-PEG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S- DAG) such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-l-0-(co-methoxy(polyethoxy) ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG- cer), or a PEG dialkoxypropylcarbamate, such as ⁇ -methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy) propy
• a PEGylated lipid suitable for the disclosure may be selected from the group consisting of PEG-DAG, DMG-PEG, PEG-PE, PEG-S-DAG, PEG-S-DMG, PEG-cer, or mPEG-N,N-ditetradecylacetamide, or a PEG-dialkyoxypropylcarbamate.
• a PEGylated lipid suitable for the disclosure may be DMG-PEG, PEG- PE, or mPEG-N,N-ditetradecylacetamide.
• a PEGylated lipid suitable for the disclosure may be DMG-PEG or PEG-PE.
• a PEGylated lipid suitable for the disclosure may be mPEG-N,N- ditetradecylacetamide.
• PEGylated lipid may be present at step a) of the method for formulating the lipid nanoparticles as disclosed herein in molar amount ranging from about 1% to about 10%, for example from about 1% to about 5%, and for example from about 1% to about 3.5% relative to the total molar amount of the lipid and ionizable lipidic compound.
• PEGylated lipid may be present in the lipid nanoparticles as disclosed herein in molar amount ranging from about 1% to about 10%, for example from about 1% to about 5%, and for example from about 1% to about 3.5% relative to the total molar amount of the lipid and lipidic compound of formula (IV), (Va) or (Vb) which may be present in the lipid nanoparticles.
• PEGylated lipid and lipidic compound of formula (IV), (Va) or (Vb) may be present in the lipid nanoparticles in a molar ratio ionizable lipidic compound to PEGylated lipid from about 70:1 to about 4:1, for example from about 40:1 to about 10:1, for example from about 35:1 to about 15:1, and for example is about 33:1 or about 14:1.
• lipid nanoparticles may comprise, further to the lipidic compound of formula (IV), (Va) or (Vb), at least one neutral lipid, at least one steroid alcohol, or an ester thereof, and at least one PEGylated lipid.
• the neutral lipids, the steroid alcohol, or ester thereof, and the PEGylated lipids may be as described herein.
• the lipid nanoparticles described herein may comprise a lipidic compound of formula (IV), (Va) or (Vb), a neutral lipid, a steroid alcohol, or an ester thereof, and a PEGylated lipid in a molar amount of about 30% to about 70% of lipidic compound, of about 0% to about 50% of neutral lipid, of 20% to about 50% of steroid alcohol or an ester thereof, and of about 1% to about 15% of PEGylated, relative to the total amount of lipid and lipidic compound.
• the lipid nanoparticles described herein may comprise a lipidic compound of formula (IV), (Va) or (Vb), a neutral lipid, a steroid alcohol or an ester thereof, and a PEGylated lipid in a molar amount of about 30% to about 60% of lipidic compound, of about 5% to about 30% of neutral lipid, of about 30% to about 48% of steroid alcohol or an ester thereof, and of about 1.5% to about 5% of PEGylated, relative to the total amount of lipid and lipidic compound.
• a lipidic compound of formula (IV), (Va) or (Vb) a neutral lipid, a steroid alcohol or an ester thereof
• PEGylated lipid in a molar amount of about 30% to about 60% of lipidic compound, of about 5% to about 30% of neutral lipid, of about 30% to about 48% of steroid alcohol or an ester thereof, and of about 1.5% to about 5% of PEGylated, relative to
• the lipid nanoparticles described herein may comprise a lipidic compound of formula (IV), (Va) or (Vb), a neutral lipid, a steroid alcohol or an ester thereof, and a PEGylated lipid in a molar amount of about 35% to about 50% of lipidic compound, of about 10% to about 16% of neutral lipid, of about 38.5% to about 46.5% of steroid alcohol or an ester thereof, and of about 1.5% of PEGylated, relative to the total amount of lipid and lipidic compound.
• a lipidic compound of formula (IV), (Va) or (Vb) a neutral lipid, a steroid alcohol or an ester thereof
• PEGylated lipid in a molar amount of about 35% to about 50% of lipidic compound, of about 10% to about 16% of neutral lipid, of about 38.5% to about 46.5% of steroid alcohol or an ester thereof, and of about 1.5% of PEGylated, relative to
• the lipid nanoparticles as disclosed herein may comprise about 35% of lipidic compound of formula (IV), (Va) or (Vb), about 16% of neutral lipid, about 46.5% of steroid alcohol, or an ester thereof, and of about 1.5% of PEGylated, relative to the total amount of lipid and lipidic compound.
• the lipid nanoparticles as disclosed herein may comprise about 50% of lipidic compound of formula (IV), (Va) or (Vb), about 10% of neutral lipid, about 38.5% of steroid alcohol or an ester thereof, and of about 1.5% of PEGylated, relative to the total amount of lipid and lipidic compound.
• the molar ratio of the lipidic compound of formula (IV), (Va) or (Vb) and of the neutral lipid, the steroid alcohol or an ester thereof, and the PEGylated lipid may be of about 35/16/46.5/1.5, of about 50/10/38.5/1.5, of about 57.2/7.1/34.3/1.4, of about 40/15/40/5, of about 50/10/35/4.5/0.5, of about 50/10/35/5, of about 40/10/40/10; of about 35/15/40/10, of about 52/13/30/5.
• the molar ratio of the lipidic compound of formula (IV), (Va) or (Vb) and of the neutral lipid, the steroid alcohol, or an ester thereof, and the PEGylated lipid may be of about 35/16/46.5/1.5 or about 50/10/38.5/1.5.
• Nucleic acids The lipid nanoparticles as disclosed herein may comprise at least one, anionic or polyanionic, therapeutic agent.
• a therapeutic agent suitable for the disclosure may be a nucleic acid.
• a nucleic acid according to the disclosure may be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA), for example RNA, for example an in vitro transcribed RNA (IVT RNA) or synthetic RNA.
• Nucleic acids according to the disclosure include genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules.
• a nucleic acid may be in the form of a molecule which is single stranded or double stranded and linear or closed covalently to form a circle.
• a nucleic can be employed for introduction into, i.e. transfection of, cells, for example, in the form of RNA which can be prepared by in vitro transcription from a DNA template. The RNA can moreover be modified before application by stabilizing sequences, capping, and polyadenylation.
• a nucleic acid may be of eukaryotic or prokaryotic origin, and for example of human, animal, plant, bacterial, yeast or viral origin and the like.
• Nucleic acids may be comprised in a vector.
• Vectors are known to the skilled person and may include plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or PI artificial chromosomes (PAC).
• the vectors include expression as well as cloning vectors.
• Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a specific host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems.
• Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments.
• the nucleic acid may be selected from a group consisting of a double stranded RNA (dsRNA); a single stranded RNA (ssRNA); a double stranded DNA (dsDNA); a single stranded DNA (ssDNA); and combinations thereof.
• the nucleic acid may be selected from a group consisting of messenger RNA (mRNA); an antisense oligonucleotide (ASO); a short interference RNA (siRNA): a self-amplifying RNA (saRNA); a micro RNA (miRNA); a small nuclear RNA (snRNA); a small nucleolar RNA (snoRNA); self-amplifying RNA (saRNA); a plasmid DNA (pDNA); closed-ended DNA (ceDNA), and combinations thereof.
• mRNA messenger RNA
• ASO antisense oligonucleotide
• siRNA short interference RNA
• saRNA self-amplifying RNA
• miRNA micro RNA
• snRNA small nuclear RNA
• snoRNA small nucleolar RNA
• saRNA self-amplifying RNA
• pDNA plasmid DNA
• ceDNA closed-ended DNA
• the nucleic acid may be selected from a group consisting of messenger RNA (mRNA); an antisense oligonucleotide (ASO); a short interference RNA (siRNA): a self-amplifying RNA (saRNA); a micro RNA (miRNA); a plasmid DNA (pDNA); and combinations thereof.
• the nucleic acid may be selected from a group consisting of messenger RNA (mRNA); a short interference RNA (siRNA): a self-amplifying RNA (saRNA); a micro RNA (miRNA); and combinations thereof.
• the nucleic acid may be a messenger RNA (mRNA).
• the nucleic acid is an mRNA.
• the nucleic acid may be a RNA encoding a protein or an enzyme.
• Such polynucleotides may be used as a therapeutic that is capable of being expressed by target cells to facilitate the production of a functional enzyme or protein.
• target cells upon the expression of at least one polynucleotide by target cells the production of a functional enzyme or protein in which a cell or an individual is deficient.
• the target cells are cells to which a composition or lipid nanoparticles as disclosed herein are to be directed or targeted.
• the target cells may comprise a specific tissue or organ.
• the target cells may be hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells (e.g., meninges, astrocytes, motor neurons, cells of the dorsal root ganglia and anterior horn motor neurons), photoreceptor cells (e.g., rods and cones), retinal pigmented epithelial cells, secretory cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, antigen presenting cells such as dendritic cells, reticulocytes, leukocytes, granulocytes and tumor cells.
• neural cells e.g., meninges, astrocytes, motor neurons, cells of the dorsal root
• RNA relates to a molecule which comprises ribonucleotide residues and for example being entirely or substantially composed of ribonucleotide residues.
• “Ribonucleotide” relates to a nucleotide with a hydroxyl group at the 2'-position of a ⁇ -D- ribofuranosyl group.
• the term includes double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, or recombinantly produced RNA.
• RNA messenger RNA
• tRNA transfer RNA
• rRNA ribosomal RNA
• siRNA stressing RNA
• miRNA miRNA
• micro RNA miRNA
• mtRNA mitochondrial RNA
• shRNA shRNA
• tmRNA transfer-messenger RNA
• vRNA viral RNA
• ssRNA single-stranded, double-stranded and/or base- paired RNA
• blunt-ended RNA or not mature and immature mRNAs, coding and non-coding RNAs, hybrid sequences or synthetic or semisynthetic sequences of oligonucleotides, modified or otherwise, and mixtures thereof.
• RNA molecules as disclosed herein also encompass monocistronic and polycistronic messenger RNAs.
• a mRNA encompasses any coding RNA molecule, which may be translated by a eukaryotic host into a protein.
• a coding RNA molecule generally refers to a RNA molecule comprising a sequence coding for a protein of interest and which may be translated by the eukaryotic host, said sequence starting with a start codon (ATG) and for example terminated by a stop codon (i.e. TAA, TAG. TGA).
• a RNA may be a naturally occurring RNA or a modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of at least one nucleotide. Such alterations can include addition of non-nucleotide material, such as to the end(s) of a RNA or internally, for example at least one nucleotide of the RNA.
• Nucleotides in RNA molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally occurring RNA.
• the RNA is a mRNA (messenger RNA).
• a mRNA may be a transcript which may be produced using DNA as template and encodes a peptide or a protein.
• mRNA typically comprises 5’Cap, a 5' non translated region (5 -UTR), a protein or a peptide coding region and a 3' non translated region (3'-UTR), and a 3’ polyA tail.
• RNA has a limited halftime in cells and in vitro.
• a mRNA is produced by in vitro transcription using a DNA template.
• the RNA may be obtained by chemical synthesis.
• the in vitro transcription methodology is known to the skilled person.
• the RNA may be in vitro synthesized in a cell-free system, using appropriate cell extracts and an appropriate DNA template.
• cloning vectors are applied for the generation of transcripts.
• the promoter for controlling transcription can be any promoter for any RNA polymerase.
• Some examples of RNA polymerases are the T7, T3, and SP6 RNA polymerases.
• a DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, for example a cDNA, and introducing it into an appropriate vector for in vitro transcription.
• the cDNA may be obtained by reverse transcription of RNA.
• cloning vectors are used for producing transcripts which generally are designated transcription vectors.
• the RNA may encode for a protein or a peptide. That is, if present in the appropriate environment, for example within a cell, such as an antigen-presenting cell, for example a dendritic cell, the RNA can be expressed to produce a protein or peptide it encodes.
• the stability and translation efficiency of a RNA may be modified as required.
• a modification of a RNA within as disclosed herein refers to any modification of RNA which is not naturally present in said RNA.
• a mRNA as disclosed herein may comprise or consist of the following general formula: [5’Cap]w - [5'UTR]x - [Gene of Interest] - [3'UTR]y - [PolyA]z wherein [5'UTR] and [3'UTR] are untranslated regions (UTR), wherein [5'UTR] contains a Kozak sequence, wherein [Gene of Interest] is any gene coding for a protein of interest, wherein [5’Cap] contains a methyl guanine nucleotide linked to mRNA via a 5′ to 5′ linkage, wherein [PolyA] is a poly(A) tail, and wherein w, x, y, and z, are identical or different, and equal to 0 or 1.
• a mRNA as disclosed herein may consist of the following general formula: [5’Cap] - [5'UTR] - [Gene of Interest] - [3'UTR] - [PolyA] wherein [5'UTR] and [3'UTR] are untranslated regions, wherein [5'UTR] contains a Kozak sequence, wherein [Gene of Interest] is any nucleic acid coding for a protein of interest, wherein [5’Cap] contains a methyl guanine nucleotide linked to mRNA via a 5′ to 5′ linkage, and wherein [PolyA] is a poly(A) tail.
• Kozak sequence refers to a sequence, which is generally a consensus sequence, occurring on eukaryotic mRNAs and which plays a major role in the initiation of the translation process.
• Kozak sequences and Kozak consensus sequences are well known in the art.
• a poly(A) tail consists of multiple adenosine monophosphates that is well known in the art.
• a poly(A) tail is generally produced during a step called polyadenylation that is one of the post-translation modifications which generally occur during the production of mature messenger RNAs; such poly(A) tail contribute to the stability and the half-life of said mRNAs, and can be of variable length.
• a poly(A) tail may be equal or longer than 10 A nucleotides, which includes equal or longer than 20 A nucleotides, which includes equal or longer than 100 A nucleotides, and for example about 120 A nucleotides.
• the [3'UTR] does not express any proteins. The purpose of the [3'UTR] is to increase the stability of the mRNA. According to a one embodiment, the a-globin UTR is chosen because it is known to be devoid of instability.
• the sequence corresponding to the gene of interest may be codon- optimized in order to obtain a satisfactory protein production within the host which is considered.
• RNA molecules as disclosed herein may be of variable length.
• RNA molecules may be short RNA molecules, for instance RNA molecules shorter than about 100 nucleotides, or long RNA molecules, for instance longer than about 100 nucleotides, or even longer than about 300 nucleotides.
• RNA such as mRNAs, may encompass synthetic or artificial RNA molecules, but also naturally occurring RNA molecules.
• a RNA molecule such as a mRNA, may encompasse the following species: (i) capped unmodified RNA molecule; (ii) capped modified RNA molecule; (iii) uncapped unmodified RNA molecule; (iv) uncapped modified RNA molecule.
• a “capped RNA molecule” refers to a RNA molecule of which the 5’end is linked to a guanosine or a modified guanosine, for example a 7- methylguanosine (m 7 G), connected to a 5’ to 5’ triphosphate linkage or analog.
• m 7 G 7- methylguanosine
• caps analogs include caps which are biologically equivalent to a 7-methylguanosine (m 7 G), connected to a 5’ to 5’ triphosphate linkage, and which can thus be also substituted without impairing the protein expression of the corresponding messenger RNA in the eukaryotic host.
• m 7 G 7-methylguanosine
• m 7 GpppN m 7 GpppG, m 7 GppspG, m 7 GppspspG, m 7 GppspspG, m 7 Gppppm 7 G, m2 7’,3’-O GpppG, m2 7’,2’-O GpppG, m2 7’,2’-O GppspsG, or m2 7’,2’- O Gppp s p s G.
• Examples of synthetic caps and/or cap analogs can be selected in a list consisting of: glyceryl, inverted deoxy abasic residue (moiety), 4',5' methylene nucleotide, 1-(beta-D- erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo-pentofuranos l nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3 '-3 '-inverted nucleotide moiety, 3 '-3 '-inverted abasic moiety, 3'- 2'-in
• caps or cap analogs include ARCA cap analogs, N1 - methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino- guanosine, LNA-guanosine, and 2-azido-guanosine.
• ARCA cap analogs N1 - methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino- guanosine, LNA-guanosine, and 2-azido-guanosine.
• synthetic caps some of the above-mentioned caps are suitable as analogs, but not others which may on the contrary hinder protein expression. Such distinction is understood by the man skilled in the art.
• the ARCA cap analog is, for instance, an example of cap analog used during in vitro transcription: it is a modified cap in which the 3’OH group (closer to m 7 G) is replaced with – OCH 3 .
• 100% of the transcripts synthesized with ARCA at the 5' end are translatable leading to a strong stimulatory effect on translation.
• RNA with a 5'-cap or 5'-cap analog may be achieved by in vitro transcription of a DNA template in the presence of said 5'-cap or 5'-cap analog, wherein said 5'- cap is co-transcriptionally incorporated into the generated RNA strand, or the RNA may be generated, for example, by in vitro transcription, and the 5'-cap may be attached to the RNA post-transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus.
• capping enzymes for example, capping enzymes of vaccinia virus.
• An “uncapped RNA molecule” refers to any RNA molecule that does not belong to the definition of a “capped RNA molecule”.
• an “uncapped mRNA” may refer to a mRNA of which the 5’end is not linked to a 7-methylguanosine, through a 5’ to 5’ triphosphate linkage, or an analog as previously defined.
• An uncapped RNA molecule such as a messenger RNA, may be an uncapped RNA molecule having a (5') ⁇ (5'), a (5') ⁇ (5'), a (5') ⁇ (5') or even a (5')OH extremity.
• RNA molecules may be respectively abbreviated as 5' ⁇ RNA; 5’ ⁇ RNA; 5’ ⁇ RNA; 5’ OH RNA.
• an uncapped RNA molecule as disclosed herein is a messenger 5' ⁇ RNA.
• RNA molecule when the RNA molecule is a single-stranded RNA molecule, it may be respectively abbreviated as 5’ppp ssRNA; 5’pp ssRNA; 5’p ssRNA; 5’OH ssRNA.
• RNA molecule when the RNA molecule is a double-stranded RNA molecule, it may be respectively abbreviated as 5’ppp dsRNA; 5’pp dsRNA; 5’p dsRNA; 5’OH dsRNA.
• an uncapped mRNA as disclosed herein is an uncapped single- stranded mRNA.
• an uncapped single-stranded mRNA may be an uncapped messenger 5’ppp ssRNA.
• the first base of said uncapped RNA molecule may be either an adenosine, a guanosine, a cytosine, or an uridine.
• an uncapped RNA molecule may be an uncapped RNA molecule having a (5’)ppp(5’), a (5’)pp(5’), a (5’)p(5’) or even a blunt-ended 5’ guanosine extremity.
• the RNA may not have uncapped 5'- triphosphates. Removal of such uncapped 5'-triphosphates can be achieved by treating RNA with a phosphatase.
• Modified and Unmodified RNA molecules The RNA may comprise further modifications.
• RNA used in the present disclosure may be an extension or truncation of the naturally occurring poly(A) tail or an alteration of the 5'- or 3'-untranslated regions (UTR) such as introduction of an UTR which is not related to the coding region of said RNA, for example, the exchange of the existing 3'-UTR with or the insertion of at least one, for example two copies of a 3'-UTR derived from a globin gene, such as alpha 2-globin, alpha 1-globin, beta-globin, for example beta-globin, and for example human beta-globin.
• UTR 5'- or 3'-untranslated regions
• a “modified RNA molecule” refers to a RNA molecule which contains at least one modified nucleotide, nucleoside or base, such as a modified purine or a modified pyrimidine.
• a modified nucleoside or base can be any nucleoside or base that is not A, U, C or G (respectively Adenosine, Uridine, Cytidine or Guanosine for nucleosides; and Adenine, Uracil, Cytosine or Guanine when referring solely to the sugar moiety).
• an “unmodified RNA molecule” refers to any RNA molecule that is not commensurate with the definition of a modified RNA molecule.
• a nucleic acid for example a RNA
• the presence of modified nucleotide may increase the stability and/or decrease cytotoxicity of the nucleic acid.
• stability of RNA relates to the half-life of RNA, that is the period of time which is needed to eliminate half of the activity, amount, or number of molecules.
• RNA refers to a RNA molecule, such as a mRNA, which contains at least one base or sugar modification as described above, and for example at least one base modification as described herein.
• a RNA suitable for the disclosure 5-methylcytidine may be substituted partially or completely, for example completely, for cytidine.
• RNA molecule may contain modified nucleotides, nucleosides or bases, including backbone modifications, sugar modifications or base modifications.
• a backbone modification in connection with the present disclosure includes modifications, in which phosphates of the backbone of the nucleotides contained in a RNA molecule as defined herein are chemically modified
• a sugar modification in connection with the present disclosure includes chemical modifications of the sugar of the nucleotides of the RNA molecule as defined herein.
• a base modification in connection with the present disclosure includes chemical modifications of the base moiety of the nucleotides of the RNA.
• nucleotide analogues or modifications are for example selected from nucleotide analogues which are suitable for transcription and/or translation of the RNA molecule in an eukaryotic cell.
• Sugar modifications may consist in replacement or modification of the 2’ hydroxy (OH) group, which can be modified or replaced with a number of different "oxy" or "deoxy” substituents.
• R H, alkyl, cycloalkyl, ary
• “Deoxy” modifications include hydrogen, amino (e.g. NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); or the amino group can be attached to the sugar through a linker, wherein the linker comprises at least one of the atoms C, N, and O
• the sugar group can also contain at least one carbon that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
• a modified RNA can include nucleotides containing, for instance, arabinose as the sugar.
• the phosphate backbone may further be modified and incorporated into the modified RNA molecule, as described herein.
• the phosphate groups of the backbone can be modified by replacing at least one of the oxygen atoms with a different substituent.
• the modified nucleosides and nucleotides can include the full replacement of an unmodified phosphate moiety with a modified phosphate as described herein.
• modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.
• Phosphorodithioates have both non-linking oxygens replaced by sulfur.
• the phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylene- phosphonates).
• the modified nucleosides and nucleotides which may be incorporated into the modified RNA molecule, as described herein, can further be modified in the nucleobase moiety.
• the nucleosides and nucleotides described herein can be chemically modified on the major groove face.
• the major groove chemical modifications can include an amino group, a thiol group, an alkyl group, or a halo group.
• nucleotide analogues/modifications are selected from base modifications selected in a list consisting of : 2-amino-6-chloropurineriboside-5'-triphosphate, 2-aminopurine-riboside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-amino-2'- deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'- fluorothymidine-5'-triphosphate, 2'-O-methyl inosine-5'-triphosphate 4-thiouridine-5'- triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5- bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'- triphosphate, 5-
• modified nucleosides may be selected from a list consisting of : pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio- pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl- uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5- taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, l- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl- pseudouridine, 2-thio-l-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl
• modified nucleosides and nucleotides include 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo- pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio- 1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1 -methyl-1-deaza- pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebula
• modified nucleosides include 2-aminopurine, 2,6- diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8- aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1- methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-d
• modified nucleosides include inosine, 1 -methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7- deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl- guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio- guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.
• the nucleotide can be modified on the major groove face and can include replacing hydrogen on C-5 of uracil with a methyl group or a halo group.
• Modified bases and/or modified RNA molecules are known in the art and are, for instance, further taught in Warren et al. (“Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA”; Cell Stem Cell; 2010).
• a modified base may be a modified purine base or a modified pyrimidine base.
• modified purine bases include modified adenosine and/or modified guanosine, such as hypoxanthine; xanthine; 7-methylguanine; inosine; xanthosine and 7-methylguanosine.
• a modified RNA or mRNA molecule corresponds to a RNA for which each nucleoside corresponding to either Uridine, Cytidine, Adenosine and/or Ribothymidine is modified.
• modified pyrimidine bases include modified cytidine and/or modified uridine, such as 5,6-dihydrouracil; pseudouridine; 5-methylcytidine; 5- hydroxymethylcytidine; dihydrouridine and 5-methylcytidine.
• a modified base as disclosed herein may be a modified uridine or cytidine, such a pseudouridine and 5-methylcytidine.
• a modified RNA corresponds to a RNA for which at least one base corresponding to either U (for Uracile), C (for Cytosine), A (for Adenine) and/or T (for Thymine) is modified.
• modified bases As example of modified bases, one may mention methyl-5 uridine (m5U), 2-thio- uridine (s2U), 2’-O-methyl-5 uridine (Ome5U), pseudouridine ( ⁇ ), methyl-1 pseudouridine (m1 ⁇ ), methyl-5 cytosine (m5C), 2’O-methyl-5 cytosine (Om5C), N6-methyl-adenosine (m6A), and N1-methyl-adenosine (m6A).
• a modified mRNA may comprise as modified bases 2’-O-methyl-5 uridine (Ome5U) or methyl-1 pseudouridine (m1 ⁇ ).
• RNA having an unmasked poly-A sequence is translated more efficiently than RNA having a masked poly-A sequence.
• poly(A) tail or "poly-A sequence” relates to a sequence of adenyl (A) residues which typically is located on the 3'-end of a RNA molecule and "unmasked poly-A sequence” means that the poly-A sequence at the 3' end of a RNA molecule ends with an A of the poly- A sequence and is not followed by nucleotides other than A located at the 3' end, i.e. downstream, of the poly-A sequence.
• a long poly-A sequence of about 120 base pairs results in an optimal transcript stability and translation efficiency of RNA. Therefore, in order to increase stability and/or expression of the RNA used according to the present disclosure, it may be modified so as to be present in conjunction with a poly-A sequence, for example having a length of 10 to 500, for example 30 to 300, for example 65 to 200 and for example 100 to 150 adenosine residues. In one embodiment the poly-A sequence has a length of approximately 120 adenosine residues. To further increase stability and/or expression of the RNA used according to the disclosure, the poly-A sequence can be unmasked.
• incorporation of a 3'-non translated region (UTR) into the 3'-non translated region of a RNA molecule can result in an enhancement in translation efficiency.
• a synergistic effect may be achieved by incorporating two or more of such 3'-non translated regions.
• the 3'- non translated regions may be autologous or heterologous to the RNA into which they are introduced.
• the 3 '-non translated region is derived from the human ⁇ -globin gene.
• RNA used according to the present disclosure may be modified within the coding region, i.e. the sequence encoding the expressed peptide or protein, for example without altering the sequence of the expressed peptide or protein, so as to increase the GC-content to increase mRNA stability and to perform a codon optimization and, thus, enhance translation in cells.
• an uncapped RNA molecule may be either a modified RNA molecule or an unmodified RNA molecule.
• a capped RNA molecule may be either a modified RNA molecule or an unmodified RNA molecule.
• a RNA molecule as disclosed herein is a messenger RNA (mRNA).
• a RNA molecule as disclosed herein is for example an uncapped messenger RNA, either in a modified or in an unmodified form.
• a RNA molecule as disclosed herein is for example a capped messenger RNA, either in a modified or in an unmodified form.
• an uncapped RNA molecule, such as a messenger RNA may also be an uncapped RNA molecule having only naturally occurring bases.
• a “naturally occurring base” relates to a base that can be naturally incorporated in vivo into a RNA molecule, such as a messenger RNA, by the host.
• a “naturally occurring base” is distinct from a synthetic base for which there would be not natural equivalent within said host.
• an uncapped messenger RNA may also be an uncapped and modified messenger RNA, and thus contain at least one modified base.
• an uncapped messenger RNA may also be an uncapped and modified messenger RNA having a (5’)ppp(5’) guanosine extremity and containing at least one modified base.
• An uncapped messenger RNA may also be an uncapped and modified messenger RNA having a (5’)ppp(5’) guanosine extremity and containing at least one pseudo-uridine and at least one 5-methylcytosine.
• a capped messenger RNA may be a messenger RNA of which the 5’end is linked to a 7-methylguanosine, or analogue, connected to a 5’ to 5’ triphosphate linkage and containing naturally occurring bases or modified bases such as pseudo-urine or 5-methyl cytosine. It is also understood that, when both modified and unmodified RNA molecules are used within one embodiment of the disclosure, they may be used either as mixtures and/or in purified forms.
• Antigens A nucleic acid contained in a lipid nanoparticle as disclosed herein may be an antigen.
• compositions as disclosed herein may be nucleic acid immunogenic composition or nucleic acid vaccines comprising at least one polynucleotide, e.g. polynucleotide constructs, which encode at least one wild type or engineered antigen.
• Antigen-containing compositions as disclosed herein may vary in their valency. Valency refers to the number of antigenic components in the composition or in the polynucleotide (e.g., RNA polynucleotide) or polypeptide.
• the immunogenic compositions are monovalent. They may also be compositions comprising more than one valence such as divalent, trivalent or multivalent compositions.
• Multivalent immunogenic compositions or vaccines may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12,13,14, 15, 16, 17, 18, 19, 20, or more antigens or antigenic moieties (e.g., antigenic peptides, etc.).
• the antigenic components may be on a single polynucleotide or on separate polynucleotides.
• Compositions as disclosed herein may be used to protect, treat or cure infection arising from contact with an infectious agent, such as bacteria, viruses, fungi, protozoa and parasites.
• Compositions as disclosed herein may be used to protect, treat or cure cancer diseases.
• a nucleic acid may encode for at least one antigen selected in the group consisting of bacterial antigens, protozoan antigens, viral antigens, fungal antigens, parasite antigens or tumour antigens.
• Bacterial antigens The bacterium described herein can be a Gram-positive bacterium or a Gram- negative bacterium.
• Bacterial antigens may be obtained from Acinetobacter baumannii, Bacillus anthracis, Bacillus subtilis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, coagulase Negative Staphylococcus, Corynebacterium diphtheria, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, enterotoxigenic Escherichia coli (ETEC), enteropathogenic E.
• Acinetobacter baumannii Bacillus anthracis
• Viral antigens may be obtained from adenovirus; Herpes simplex, type 1 ; Herpes simplex, type 2; encephalitis virus, papillomavirus, Varicella-zoster virus; Epstein-barr virus; Human cytomegalovirus; Human herpesvirus, type 8; Human papillomavirus; BK virus; JC virus; Smallpox; polio virus, Hepatitis B virus; Human bocavirus; Parvovirus B19; Human astrovirus; Norwalk virus; coxsackievirus; hepatitis A virus; poliovirus; rhinovirus; Severe acute respiratory syndrome virus; Hepatitis C virus; yellow fever virus; dengue virus; West Nile virus; Rubella virus; Hepatitis E virus; Human immunodeficiency virus (HIV); Influenza virus, type A or B; Guanarito virus; Junin virus; Lassa virus; Machupo virus; Sabia virus; Crimean-Congo hemor
• the antigen is from a strain of Influenza A or Influenza B virus or combinations thereof.
• the strain of Influenza A or Influenza B may be associated with birds, pigs, horses, dogs, humans or non-human primates.
• the nucleic acid may encode a hemagglutinin protein or a fragment thereof.
• the hemagglutinin protein may be H1, H 2 , H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18, or a fragment thereof.
• the hemagglutinin protein may or may not comprise a head domain (HA1). Alternatively, the hemagglutinin protein may or may not comprise a cytoplasmic domain.
• the hemagglutinin protein is a truncated hemagglutinin protein.
• the truncated hemagglutinin protein may comprise a portion of the transmembrane domain.
• the virus may be selected from the group consisting of H1N1, H3N2, H7N9, H5N1 and H10N8 virus or a B strain virus.
• the antigen is from a coronavirus such as SARS-Cov-1 virus, SARS-Cov-2 virus, or MERS-Cov virus.
• Fungal antigens may be obtained from Ascomycota (e.g., Fusarium oxysporum, Pneumocystis jirovecii, Aspergillus spp., Coccidioides immitis/posadasii, Candida albicans), Basidiomycota (e.g., Filobasidiella neoformans, Trichosporon), Microsporidia (e.g., Encephalitozoon cuniculi, Enterocytozoon bieneusi), and Mucoromycotina (e.g., Mucor circinelloides, Rhizopus oryzae, Lichtheimia corymbifera).
• Ascomycota e.g., Fusarium oxysporum, Pneumocystis jirovecii, Aspergillus spp., Coccidioides immitis/posadasii, Candida albicans
• Basidiomycota e
• Protozoan antigens may be obtained from Entamoeba histolytica, Giardia lambila, Trichomonas vaginalis, Trypanosoma brucei, T. cruzi, Leishmania donovani, Balantidium coli, Toxoplasma gondii, Plasmodium spp., and Babesia microti.
• Parasitic antigens may be obtained from Acanthamoeba, Anisakis, Ascaris lumbricoides, botfly, Balantidium coli, bedbug, Cestoda, chiggers, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, hookworm, Leishmania, Linguatula serrata, liver fluke, Loa loa, Paragonimus, pinworm, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, mite, tapeworm, Toxoplasma gondii, Trypanosoma, whipworm, Wuchereria bancrofti.
• an antigen may be a tumor antigen, i.e., a constituent of cancer cells such as a protein or a peptide expressed in a cancer cell.
• tumor antigen relates to proteins that are under normal conditions specifically expressed in a limited number of tissues and/or organs or in specific developmental stages and are expressed or aberrantly expressed in at least one tumor or cancer tissue.
• Tumor antigens include, for example, differentiation antigens, for example cell type specific differentiation antigens, i.e., proteins that are under normal conditions specifically expressed in a certain cell type at a certain differentiation stage and germ line specific antigens.
• a tumor antigen is presented by a cancer cell in which it is expressed.
• tumor antigens include the carcinoembryonal antigen, a 1-fetoprotein, isoferritin, and fetal sulphoglycoprotein, cc2-H- ferroprotein and ⁇ -fetoprotein.
• Other examples for tumor antigens that may be useful in the present disclosure are p53, ART-4, BAGE, beta-catenin/m, Bcr-abL CAMEL, CAP-1 , CASP-8, CDC27/m, CD 4/m, CEA, the cell surface proteins of the claudin family, such as CLAUDIN-6, CLAUDIN-18.2 and CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1 , G250, GAGE, GnT-V, Gapl OO, HAGE, HER-2/neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR/F
• Adjuvants Nucleic acid containing compositions or lipid nanoparticles as disclosed herein may further comprise, or may be co-administered with, an adjuvant or an immune potentiator.
• Adjuvants useful in the present disclosure may include, but are not limited to, natural or synthetic adjuvants. They may be organic or inorganic.
• Adjuvants may be selected from any of the classes (1) mineral salts, e.g., aluminium hydroxide and aluminium or calcium phosphate gels; (2) emulsions including: oil emulsions and surfactant based formulations, e.g., microfluidized detergent stabilized oil-in-water emulsion, purified saponin, oil-in- water emulsion, stabilized water-in-oil emulsion; (3) particulate adjuvants, e.g., virosomes (unilamellar liposomal vehicles incorporating influenza haemagglutinin), structured complex of saponins and lipids, polylactide co-glycolide (PLG); (4) microbial derivatives; (5) endogenous human immunomodulators; and/or (6) inert vehicles, such as gold particles; (7) microorganism derived adjuvants; (8) tensioactive compounds; (9) carbohydrates; or combinations thereof.
• mineral salts e
• adjuvants may include, without limitation, cationic liposome-DNA complex JVRS-100, aluminum hydroxide vaccine adjuvant, aluminum phosphate vaccine adjuvant, aluminum potassium sulfate adjuvant, alhydrogel, ISCOM(s)TM, Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, CpG DNA Vaccine Adjuvant, Cholera toxin, Cholera toxin B subunit, Liposomes, Saponin Vaccine Adjuvant, DDA Adjuvant, Squalene-based Adjuvants, Etx B subunit Adjuvant, IL-12 Vaccine Adjuvant, LTK63 Vaccine Mutant Adjuvant, TiterMax Gold Adjuvant, Ribi Vaccine Adjuvant, Montanide ISA 720 Adjuvant, Corynebacterium- derb/ed P40 Vaccine Adjuvant, MPLTM Adjuvant
• compositions as disclosed herein or the lipid nanoparticles as disclosed herein encapsulating at least one nucleic acid may also be used for treating individuals deficient in a protein. Therefore, the lipid nanoparticles may be used in a method for treating individuals deficient in a protein comprising administering lipid nanoparticles comprising at least one nucleic acid, for example a mRNA, wherein the nucleic acid encodes a functional protein corresponding to the protein which is deficient in the individual.
• a functional protein is produced following expression of the nucleic acid by a target cell.
• the disclosure also relates to methods of intracellular delivery of nucleic acids that are capable of correcting existing genetic defects and/or providing beneficial functions to at least one target cell.
• compositions and nucleic acids of the present disclosure transfect that target cell and the nucleic acids (e.g., mRNA) can be translated into the gene product of interest (e.g., a functional protein or enzyme) or can otherwise modulate or regulate the presence or expression of the gene product of interest.
• the compositions and methods provided herein are useful in the management and treatment of a large number of diseases, for example diseases which result from protein and/or enzyme deficiencies. Individuals suffering from such diseases may have underlying genetic defects that lead to the compromised expression of a protein or an enzyme, including, for example, the non-synthesis of the protein, the reduced synthesis of the protein, or synthesis of a protein lacking or having diminished biological activity.
• the nucleic acids may encode full length antibodies or smaller antibodies (e.g., both heavy and light chains) to confer immunity to a subject.
• the compositions as described herein encode antibodies that may be used to transiently or chronically effect a functional response in subjects.
• the mRNA nucleic acids as described herein may encode a functional monoclonal or polyclonal antibody, which upon translation (and as applicable, systemic excretion from the target cells) may be useful for targeting and/or inactivating a biological target (e.g., a stimulatory cytokine such as tumor necrosis factor).
• the mRNA nucleic acids as described herein may encode, for example, functional anti-nephritic factor antibodies useful for the treatment of membranoproliferative glomerulonephritis type II or acute hemolytic uremic syndrome, or alternatively may encode anti-vascular endothelial growth factor (VEGF) antibodies useful for the treatment of VEGF-mediated diseases, such as cancer.
• VEGF vascular endothelial growth factor
• the disclosure relates to pharmaceutical compositions, such as immunogenic compositions.
• the lipid nanoparticles as disclosed herein comprising a therapeutic agent, such as a nucleic acid may be administered as pharmaceutical compositions.
• compositions of the present disclosure comprise lipid nanoparticles as disclosed herein and at least one pharmaceutically acceptable carrier, diluent or excipient.
• pharmaceutical compositions suitable for the disclosure may comprise (i) at least one nucleic acid and at least one lipidic compound as disclosed herein, or (ii) at least one composition as described herein, or (iii) at least one lipid nanoparticle as described herein, and at least one pharmaceutically acceptable excipient.
• a pharmaceutical composition may be an immunogenic composition.
• An immunogenic composition as disclosed herein may comprise at least one lipid nanoparticle as described herein, wherein the nucleic acid contained thereof encodes for at least one antigen.
• an immunogenic composition may comprise at least one adjuvant as described herein.
• the disclosure relates to a composition comprising at least one lipid nanoparticle as described herein for use as a medicament. Such a medicament may be used for the prevention and/or treatment of a disease as indicated herein.
• the disclosure relates to a composition comprising at least one lipid nanoparticle as described herein, for use in a therapeutic method for preventing and/or treating a disease selected in a group consisting of infectious diseases, allergies, autoimmune diseases, rare blood disorders, rare metabolic diseases, rare neurologic diseases, and tumour or cancer diseases, and for example as herein described.
• a composition comprising at least one lipid nanoparticle as herein described may be for use as an immunogenic composition.
• Immunogenic compositions as disclosed herein may be used in the prevention and/or treatment of an infectious diseases as indicated herein. They may contain at least one nucleic acid encoding for at least one antigen as herein described.
• the lipidic compound of formula (IV), (Va) or (Vb) may be present in a pharmaceutical or immunogenic composition in an amount which is effective to form lipid nanoparticles and deliver the therapeutic agent, for example a nucleic acid, for treating a specific disease or condition of interest. Appropriate concentrations and dosages can be readily determined by one skilled in the art.
• compositions as disclosed herein may be carried out via any of the accepted modes of administration of compositions for serving similar utilities.
• the compositions as disclosed herein may be formulated into preparations in solid, semi-solid, liquid forms, such as powders, solutions, suspensions or injections.
• Typical routes of administering such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, intranasal.
• parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques.
• a composition as disclosed herein may be administered by transdermal, subcutaneous, intradermal or intramuscular route.
• compositions as disclosed herein are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000).
• the compositions may contain at least one inert diluent or carrier.
• the composition may be in the form of a liquid, for example, a solution, an emulsion or a suspension.
• the liquid may be for delivery by injection.
• compositions intended to be administered by injection may contain at least one of: a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
• the liquid compositions as disclosed herein may include at least one of: sterile diluents such as water for injection, saline solution, such as physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose; agents to act as cryoprotectants such as suc
• the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
• An injectable pharmaceutical composition is for example sterile.
• the pharmaceutical and immunogenic compositions as disclosed herein may be prepared by methodology well known in the pharmaceutical art.
• a pharmaceutical composition intended to be administered by injection can be prepared by combining the lipid nanoparticles as disclosed herein with sterile, distilled water or other carrier so as to form a solution.
• a surfactant may be added to facilitate the formation of a homogeneous solution or suspension.
• compositions as disclosed herein are administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific therapeutic agent employed; the metabolic stability and length of action of the therapeutic agent; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the specific disorder or condition; and the subject undergoing therapy.
• Compositions as disclosed herein may also be administered simultaneously with, prior to, or after administration of at least one other therapeutic agent.
• Such combination therapy includes administration of a single pharmaceutical dosage formulation of a composition as disclosed herein and at least one additional active agent, as well as administration of the composition as disclosed herein and each active agent in its own separate pharmaceutical dosage formulation.
• compositions as disclosed herein and at least one additional active agent can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially; combination therapy is understood to include all these regimens.
• Methods of treatment also relates to a method of preventing and/or treating a disease in an individual in need thereof, wherein the method comprises administering an effective amount of at least one lipid nanoparticle as disclosed herein, to said individual.
• a composition containing the LNPs as disclosed herein may be for use in a therapeutic method for preventing and/or treating infectious diseases, allergies, autoimmune diseases, rare blood disorders, rare metabolic diseases, rare neurologic diseases, and tumour or cancer diseases.
• the disclosure also relates to a use of at least one lipid nanoparticle as disclosed herein for the manufacture of a medicament for preventing and/or treating infectious diseases, allergies, autoimmune diseases, rare blood disorders, rare metabolic diseases, rare neurologic diseases, and tumour or cancer diseases.
• infectious diseases such as viral infectious diseases, bacterial infectious diseases, fungal or parasitic infectious diseases.
• Diseases also concerned by the disclosure may be cancer or tumour diseases.
• Viral infectious diseases may be acute febrile pharyngitis, pharyngoconjunctival fever, epidemic keratoconjunctivitis, infantile gastroenteritis, Coxsackie infections, infectious mononucleosis, Burkitt lymphoma, acute hepatitis, chronic hepatitis, hepatic cirrhosis, hepatocellular carcinoma, primary HSV-1 infection (e.g., gingivostomatitis in children, tonsillitis and pharyngitis in adults, keratoconjunctivitis), latent HSV-1 infection (e.g., herpes labialis and cold sores), primary HSV-2 infection, latent HSV-2 infection, aseptic meningitis, infectious mononucleosis, Cytomegalic inclusion disease, Kaposi sarcoma, multicentric Castleman disease, primary effusion lymphoma, AIDS, influenza, Reye syndrome, measles, postinfectious encephal
• the disease is influenza, a Respiratory Syncytial Virus (RSV) infection, or Covid-19, and for example is influenza.
• Bacterial infectious diseases may be such as abscesses, actinomycosis, acute prostatitis, aeromonas hydrophila, annual ryegrass toxicity, anthrax, bacillary peliosis, bacteremia, bacterial gastroenteritis, bacterial meningitis, bacterial pneumonia, bacterial vaginosis, bacterium-related cutaneous conditions, bartonellosis, BCG-oma, botryomycosis, botulism, Brazilian purpuric fever, Brodie abscess, brucellosis, Buruli ulcer, campylobacteriosis, caries, Carrion's disease, cat scratch disease, cellulitis, chlamydia infection, cholera, chronic bacterial prostatitis, chronic recurrent multifocal osteomyelitis, clostridial necrotizing
• Parasitic infectious diseases may be amoebiasis, giardiasis, trichomoniasis, African Sleeping Sickness, American Sleeping Sickness, leishmaniasis (Kala-Azar), balantidiasis, toxoplasmosis, malaria, acanthamoeba keratitis, and babesiosis.
• Fungal infectious diseases may be aspergilloses, blastomycosis, candidasis, coccidioidomycosis, cryptococcosis, histoplasmosis, mycetomas, paracoccidioidomycosis, and tinea pedis.
• fungi can attack eyes, nails, hair, and especially skin, the so-called dermatophytic fungi and keratinophilic fungi, and cause a variety of conditions, of which ringworms such as athlete's foot are common.
• Fungal spores are also a major cause of allergies, and a wide range of fungi from different taxonomic groups can evoke allergic reactions in some people.
• cervical carcinoma cervical cancer
• Diseases for which the present disclosure can be useful as a therapeutic intervention include diseases such as SMN1-related spinal muscular atrophy (SMA); amyotrophic lateral sclerosis (ALS); GALT-related galactosemia; Cystic Fibrosis (CF); SLC3A1-related disorders including cystinuria; COL4A5-related disorders including Alport syndrome; galactocerebrosidase deficiencies; X-linked adrenoleukodystrophy and adrenomyeloneuropathy; Friedreich's ataxia; Pelizaeus-Merzbacher disease; TSC1 and TSC2- related tuberous sclerosis; Sanfilippo B syndrome (MPS IIIB); CTNS-related cystinosis; the FMR 1 -related disorders which include Fragile X syndrome, Fragile X-Associated Tremor/Ataxia Syndrome and Fragile X Premature Ovarian Failure Syndrome; Prader-Willi syndrome; hereditary hemorrhagic telan
• the nucleic acids, and for example mRNA, of the present disclosure may encode functional proteins or enzymes.
• the compositions of the present disclosure may include mRNA encoding erythropoietin (EPO), ⁇ 1-antitrypsin, carboxypeptidase N, alpha galactosidase (GLA), ornithine carbamoyltransferase (OTC), or human growth hormone (hGH).
• EPO erythropoietin
• GLA alpha galactosidase
• OTC ornithine carbamoyltransferase
• hGH human growth hormone
• the disclosure relates to methods of transfecting at least one isolated target cell with a nucleic acid, wherein said method comprises contacting the at least one target cell with an effective amount of at least one nucleic acid and at least one lipid nanoparticle as above described, such that the at least one target cell are transfected with said nucleic acid.
• Target cells include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells (e.g., meninges, astrocytes, motor neurons, cells of the dorsal root ganglia and anterior horn motor neurons), photoreceptor cells (e.g., rods and cones), retinal pigmented epithelial cells, secretory cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, antigen presenting cells such as dendritic cells, reticulocytes, leukocytes, granulocytes and tumor cells.
• neural cells e.g., meninges, astrocytes, motor neurons, cells of the
• the cells targeted may be spleen, liver, lung, heart and kidney cells. In another embodiment, the cells targeted may be spleen and kidney cells, and for example may be spleen cells.
• lipid nanoparticles or compositions as disclosed herein which allow avoiding hepatic clearance may be of particular interest. Following transfection of at least one target cell by, for example, the nucleic acid encapsulated in the lipid nanoparticles, the production of a polypeptide or a protein encoded by such nucleic acid may be for example stimulated and the capability of such target cells to express the nucleic acid and produce, for example, a polypeptide or protein of interest is enhanced.
• the disclosure relates to methods of producing a polypeptide in at least one target cell, wherein said method comprises contacting the at least one target cell with an effective amount of at least one nucleic acid encoding said polypeptide and at least one lipid nanoparticle as herein described, such that the at least one target cell are transfected with the nucleic acid operably encoding said polypeptide.
• NMR spectra were obtained using the commercial software NMRnotebook.
• HRMS High-resolution mass spectra
• LCMS Low-resolution mass spectra
• Example 1 Synthesis of 2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy] propoxy] ethoxy] ethoxy] ethoxy]-2-oxo-ethyl]-[2-[2-(2-methoxyethylammonio) acetyl] oxyethyl] ammonium;2,2,2-trifluoroacetate (compound of formula (III) in the disclosure as trifluoroacetate salt - DOG-CLEAVE) and its conversion as a dihydrochloride salt
• the compound 14 is prepared according to the following schema of synthesis:
• the crude product was purified by column chromatography on 120 g SI60 (n-Hexane/ethyl acetate 0% ⁇ 40% on 10 VC then 40% on 11 VC) to give the coupling 2-[(2-benzyloxy-2-oxo-ethyl)-tert-butoxycarbonyl-amino]ethyl 2-[tert- butoxycarbonyl(2-methoxyethyl)amino]acetate as a colorless oil (7.92 g, 70% Yield).
• Step (2) To a mixture of 2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethanol (15.6 g, 0.0357 mol) and N,N-diethylethanamine (7.23 g, 0.0715 mol) in DCM (300 mL) was added methanesulfonyl chloride (4.91 g, 0.0429 mol) slowly at 0 ° C. The mixture was stirred overnight at room temperature. Water (150 mL) and CH 2 Cl2 (300 mL) were added to the solution, and the mixture was transferred to a separatory funnel. The mixture was shaken, the layers were separated, and the organic layer was collected.
• Step (4) To a mixture of [2-[2-[2-[2-[2,3-bis[(Z)-octadec-9- enoxy]propoxy]ethoxy]ethoxy]ethoxy- diphenyl-methyl]benzene (1.0 g, 0.99 mmol) in THF (10 mL)/ MeOH (10 mL) was added Toluene-4-sulfonic acid (0.188 g, 0.99 mmol) and stirred overnight at room temperature. Et 3 N (0.3 mL) was added to the reaction mixture and concentrated.
• Step (5) In a 50 mL round bottom flask, 2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2- methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (200 mg, 0.46 mmol), 2-[2-[2-[2-[2,3- bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethanol (354 mg, 0.46 mmol) and N,N-dimethylpyridin-4-amine (84.4 mg, 0.69 mmol) were dissolved in DCM-Anhydrous (10 mL) in presence of 4-methylmorpholine (69.8 mg, 0.69 mmol).
• Step (6) To a mixture of 2-[[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9- enoxy]propoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetate (0.32 g, 0.27 mmol) in DCM (5 mL) was added TFA (0.308 g, 2.7 mmol) at room temperature. The mixture was stirred at ambient temperature for 16 h.
• Example 3 Synthesis of 2-[[2-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9- enoxy]propoxy]ethoxy]ethoxy]ethoxy]-2-oxoethyl]amino]ethyl (2S)-2-amino-3- methoxy-propanoate; hydrochloride (compound (VI)) Compound (VI) Compound (VI) was synthesized based on the chemistry shown in Scheme (5) represented on Figure 4. Compound (VI-A) was synthesized based on the chemistry shown in Scheme (6).
• Step (2) To the solution of 2-[(2-benzyloxy-2-oxo-ethyl)-tert-butoxycarbonyl-amino]ethyl (2S)-2-(tert-butoxycarbonylamino)-3-methoxy-propanoate(8.51 g, 16.7 mmol) in ethyl acetate (90 ml) was added palladium (10%, 1.77 g, 1.67 mmol). The mixture was stirred at room temperature under hydrogen atmosphere for 16h. LCMS showed the start material was converted into the product. The mixture was filtered through celite, wash with ethyl acetate.
• Step (2) A solution of 2-[[2-[2-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9- enoxy]propoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl (2S)-2-(tert-butoxycarbonylamino)-3-methoxy-propanoate(200 mg, 0.171 mmol) in 3 M HCl in ethyl acetate (2 ml) was stirred for 16 hrs at room temperature.
• Step (2) To a solution of 2-[2-(2-aminoethoxy)ethoxy]ethanol (1.0 g, 6.7 mmol) and imidazole (1.08 g, 15.4 mmol) in CH 2 Cl 2 (20 mL) was added TBDPSCl (2.18 g, 7.71 mmol). The reaction was stirred for 18 hrs at room temperature. The mixture was diluted with CH 2 Cl 2 (30 mL) and washed the resulting mixture with brine. The organic layer was dried over Na 2 SO 4 , filtered and concentrated.
• Step (4) The mixture of 2,3-bis[(Z)-octadec-9-enoxy]propyl N-[2-[2-[2-[tert- butyl(diphenyl)silyl]oxyethoxy]ethoxy]ethyl]carbamate (500 mg, 0.47 mmol) and TBAF (1 M in THF, 3 mL) was stirred at 25°C for 2 h. TLC (ethyl acetate/petroleum ether 1/1) indicated that the starting material was consumed and a new spot was observed.
• Step (6) A mixture of 2-[[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9- enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert- butoxycarbonyl-amino]ethyl 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetate (0.6 g, 0.334 mmol) in DCM (5 ml) was added TFA (0.381 g, 3.34 mmol). The mixture was stirred 3 h at ambient temperature.
• Step (3) 2-[[2-[2-[2-[2-(2-benzyloxy-2-oxo-ethoxy)ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert- butoxycarbonyl-amino]ethyl 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetate (1.2 g, 1.68 mmol) and Pd/C (0.5 g) were mixed with EtOAc (30 mL) and attached to a hydrogenation apparatus. The system was evacuated and then refilled with hydrogen. The mixture was stirred at room temperature overnight.
• Step (4) In a 50 mL round bottom flask, 2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2- methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (400 mg, 0.64 mmol), 2,3-bis[(Z)- octadec-9-enoxy]propan-1-ol (380 mg, 0.64 mmol) and N,N-dimethylpyridin-4-amine (117 mg, 0.96 mmol) were dissolved in DCM-Anhydrous (15 mL) in presence of 4-methylmorpholine (97.2 mg, 0.96 mmol).
• Step (5) To a mixture of 2-[[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]-2-oxo- ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl 2-[tert- butoxycarbonyl(2-methoxyethyl)amino]acetate (0.45 g, 0.375 mmol) in DCM (5 mL) was added HCl in EtOAc (5 mL, 3 mol/L). The mixture was stirred 3 hrs at room temperature.
• Example 12 Synthesis of nonyl 8-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2- methoxyethylamino)acetyl]oxyethylamino]acetyl]oxyethoxy]ethoxy]ethoxy]-2-(8- nonoxy-8- oxo-octoxy)propoxy]octanoate;2,2,2-trifluoroacetic acid (compound (XV))
• Compound (XV) was synthesized based on the chemistry shown in Scheme (9) presented on Figure 7.
• Step (2) To the solution of (2,2-dimethyl-1,3-dioxolan-4-yl)methanol (24.4 g, 175 mmol) in THF (500 mL) was added NaH (14 g, 351 mmol) and the mixture was heated to reflux for 15 min. Then the reaction was cooled to room temperature and 2-[2-[2-(2- benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (69.1 g, 175 mmol) was added under nitrogen and the reaction was heated at 80 °C for 24 h. TLC indicated that the starting material was consumed. The reaction was quenched with water and extracted with ethyl acetate.
• Step (3) The mixture of 4-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2,2- dimethyl-1,3-dioxolane (54.4 g, 123 mmol) in AcOH (200 mL) and H 2 O (200 mL) was stirred at room temperature for 18 h. TLC (EA/PE 1/1, SM Rf: 0.5; product, Rf: 0.1) indicated that all the starting materials was consumed. The solvent was removed under vacuum and azeotroped with toluene several times.
• Step (6) 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(8- oxooctoxy)propoxy]octanoic acid (10 g, 8 mmol) was dissolved in t-BuOH : H2O ( 3:1, 160 mL), containing NaH2PO4.2H2O (3.73 g, 24 mmol), 2-methy-2-butene (40 mL) and sodium chlorite (2.71 mg, 24 mmol). The reaction was stirred for 2 h at rt and LCMS indicated that the starting material was consumed. The reaction mixture was diluted with ethyl acetate.
• Step (7) To the solution of 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7- carboxyheptoxy)propoxy]octanoic acid (10 g, 14.8 mmol) and 1-Nonanol (5.12 g, 35.5 mmol) in dry dichloromethane (200 mL) under nitrogen were added N,N-Diisopropylethylamine (11.5 g, 88.7 mmol), DMAP (0.722 g, 5.91 mmol) and EDCI (7.37 g, 38.4 mmol). The mixture was stirred at room temperature for 18 h.
• Step (8) To the solution of nonyl 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2- (8-nonoxy-8-oxo-octoxy)propoxy]octanoate (5 g, 5.31 mmol) in ethyl acetate (100 mL) was added Pd/C (1.13 g, 20% wt/wt). The mixture was stirred at room temperature under hydrogen for 18 h. TLC (ethyl acetate/petroleum ether 1/1) indicated that the starting material was consumed.
• TLC ethyl acetate/petroleum ether 1/1
• Step (9) Nonyl 8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo- octoxy)propoxy]octanoate (330 mg, 0.39 mmol), 2-[tert-butoxycarbonyl-[3-[tert- butoxycarbonyl(2-methoxyethyl)amino]-2-oxo-propyl]amino]acetic acid (199 mg, 0.467 mmol) and N,N-dimethylpyridin-4-amine (71.4 mg, 0.58 mmol) were dissolved in DCM-Anhydrous (15 mL) in presence of 4-methylmorpholine (59.1 mg, 0.58 mmol).
• Example 14 Synthesis of [(Z)-non-2-enyl] 8-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2- methoxyethylamino)acetyl]oxyethylamino]acetyl]oxyethoxy]ethoxy]ethoxy]-2-[8- [(Z)-non-2- enoxy]-8-oxo-octoxy]propoxy]octanoate;2,2,2-trifluoroacetic acid (compound (XVII)) C Compound (XVII) was synthesized based on the chemistry shown in Scheme (10) presented on Figure 8.
• Step (2) To a mixture of 2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethanol (33 g, 0.0756 mol) and N,N-diethylethanamine (15.3 g, 0.151 mol) in DCM (600 mL) was added methanesulfonyl chloride (10.4 g, 0.0907 mol) slowly at 0 o C. The mixture was stirred overnight at room temperature. CH 2 Cl 2 (400 mL) were added to the solution, and the mixture was washed with diluted HCl (1M,1000 mL).The mixture was shaken, the layers were separated, and the organic layer was collected.
• Step (3) To a suspension of NaH (9.09 g, 60% in oil, 227 mmoles) in 300 ml of anhydrous DMF was added 3-trityloxypropane-1,2-diol (20 g, 56.8 mmoles) . The mixture was heated at 80 o C for 15 minutes and cooled to room temperature.9-bromonon-1-ene (30 g, 142 mmoles) in 10 ml of anhydrous DMF was added dropwise to the mixture which was then heated under 80°C for 18 h. After cooling to RT, 500 ml of H2O were added to destroy remaining NaH. The organic phase was extracted with 3 x 250 ml ethyl acetate.
• the extract was washed successively with 2 x150 ml of 1N HCl, 2 x 150 ml of 5% (w/v) NaHCO3 and 150 ml of brine and dried on Na2SO4.
• the solvent was evaporated under reduced pressure and the resulting oil was purified on a silica gel column eluted with petroleum ether/ethyl acetate (2% to 5% ethyl acetate in petroleum ether) to yield a colorless oil (12 g; yield 34.4%).
• Step (4) To a solution of [2,3-bis(non-8-enoxy)propoxy-diphenyl-methyl]benzene (10 g, 16.3 mmol) in methanol / THF (150 mL, 1/1 v/v) was added p-Toluenesulfonic acid (14 g, 81.5 mmol) in one portion at room temperature and the mixture was stirred at room temperature for 18 h. TLC (4% ethyl acetate in petroleum ether) indicated that the starting material was disappeared completely.10 mL triethylamine was added to quench the reaction and the solvent was removed under vacuum.
• Step (7) 8-[2-(8-oxooctoxy)-3-[2-[2-[2-(2- trityloxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]octanoicacid (6.98 g, 3.58 mmol) was dissolved in t-BuOH : H 2 O ( 3:1, 120 mL), containing NaH 2 PO 4 (1.28 g, 10.8 mmol), 2-methy-2-butene (11 mL) and sodium chlorite (0.972 g, 10.8 mmol). The reaction was stirred for 2 h at rt and LCMS indicated that the starting material was consumed. The reaction mixture was diluted with H 2 O.
• Step (8) To the solution of 8-[2-(7-carboxyheptoxy)-3-[2-[2-[2-(2- trityloxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]octanoic acid(6.161 g, 7.75 mmol) and (Z)- non-2-en-1-ol (2.65 g, 18.6 mmol) in dry dichloromethane (120 mL) then added DIPEA (6.01 g, 46.5 mmol), DMAP (0.379 g, 0.31 mmol) and under ice bath added EDCI(3.86 g, 20.1 mmol). The mixture was stirred at room temperature for 18 hrs.
• Step (9) To a solution of [(Z)-non-2-enyl] 8-[2-[8-[(Z)-non-2-enoxy]-8-oxo-octoxy]-3-[2-[2-[2- (2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]octanoate (0.920 g, 0.882 mmol) in methanol / THF (60mL, 1/1 v/v) was added p-Toluenesulfonic acid (0.839, 4.41 mmol) in one portion at room temperature and the mixture was stirred at room temperature for 2 h.
• Step (10) To a solution of[(Z)-non-2-enyl] 8-[3-[2-[2-[2-(2- hydroxyethoxy)ethoxy]ethoxy]-2-[8-[(Z)-non-2-enoxy]-8-oxo- octoxy]propoxy]octanoate(0.44 g, 0.549 mmol) in dry DCM (15 mL) was added 2-[tert- butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2- methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (0.239 g, 0.549 mmol), 4- methylmorpholine (0.084 g, 0.824 mmol) and DMAP (0.101 g, 0.824 mmol) at room temperature.
• Step (2) To a solution of (7R,11R)-3,7,11,15-tetramethylhexadecan-1-ol (23.5 g, 78.7 mmol) in DCM (300 ml) was added Triethylamine (15.9 g, 157 mmol) and Methanesulfonyl chloride (13.5 g, 118 mmol) at 0°C. The mixture was stirred at 25°C for 14 hr.
• Step (4) To a solution of [[3-non-8-enoxy-2-[(7R,11R)-3,7,11,15- tetramethylhexadecoxy]propoxy]-diphenylmethyl]benzene (3.5 g, 4.73 mmol) in MeOH/THF (30 ml 1:1) was added Toluene-4-sulfonic acid (4.50 g, 23.7 mmol) . The mixture was stirred at 25°C for 14 hr. The mixture was concentrated and dealt with EA (150 ml), washed with water (150 ml x2), NaCl sat.aq (150 ml) and dried over Na 2 SO 4 .
• Step (5) To a mixture of 2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethanol (33 g, 0.0756 mol) and N,N-diethylethanamine (15.3 g, 0.151 mol) in DCM (600 mL) was added methanesulfonyl chloride (10.4 g, 0.0907 mol) slowly at 0°C. The mixture was stirred overnight at room temperature. CH 2 Cl 2 (400 mL) were added to the solution, and the mixture was washed with diluted HCl(1M,1000mL). The mixture was shaken, the layers were separated, and the organic layer was collected.
• Step (7) To a solution of [2-[2-[2-[2-[3-non-8-enoxy-2-[(7R,11R)-3,7,11,15- tetramethylhexadecoxy]propoxy]ethoxy]ethoxy]ethoxy-diphenyl-methyl]benzene (2.11 g, 2.3 mmol) in ACN/CCl4/H2O (5ml/5ml/5ml) was added NaIO4 (1.97 g, 9.2 mmol) and ruthenium(III) chloride (47.8 mg, 0.23 mmol). The mixture was stirred at 25°C for 14 hrs.
• Step (8) To a solution of (Z)-non-2-en-1-ol (326mg, 2.3 mmol) in DCM (20 ml) was added 8- [2-[(7R,11R)-3,7,11,15- tetramethylhexadecoxy]-3-[2-[2-[2-(2- trityloxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]octanoic acid (1.78 g, 1.9 mmol), DIEA (739 mg, 5.72 mmol), 4-Dimethylaminopyridine (46.4 mg, 0.38 mmol ) and EDC HCl (548 mg, 2.86 mmol). The mixture was stirred at 26°C for 18 hrs.
• Step (2) 2,3-bis[(Z)-octadec-9-enoxy]propanal (1.48 g, 2.5 mmol) was dissolved in t-BuOH : H 2 O ( 3:1, 20 mL), containing NaH2PO4.2H 2 O (1.17g, 7.51 mmol), 2-methy-2-butene (5.26 ml) and sodium chlorite (679 mg, 7.51 mmol).
• the reaction was stirred for 1 h at rt and was diluted with ethyl acetate (100 ml) washed with water(100 ml x2). The aqueous layer was extracted with ethyl acetate (100 ml).
• Step (3) 2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl methanesulfonate (5 g, 8 mmol) was added to octan-1- amine (20 ml), and the mixture was stirred at 80°C for 18 hrs. LCMS showed the SM was consumed and the product was formed. The mixture was dealt with EA (300 ml), washed with water (300 mlx2 ), NaCl sat.aq (300 ml) and dried over Na2SO4.
• Step (5) To a solution of 2,3-bis[(Z)-octadec-9-enoxy]-N-octyl-N-[2-[2-[2-(2- trityloxyethoxy)ethoxy]ethoxy]ethyl]propanamide (1079 mg, 0.9 mmol) in THF/MeOH (10 ml, 1/1) was added Toluene-4-sulfonic acid (869 mg, 4.57 mmol). The mixture was stirred at 25°C for 2 hr.
• Step (6) To a solution of N-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]-2,3- bis[(Z)-octadec-9-enoxy]-N-octylpropanamide (350 mg, 0.3 mmol) in DCM (5 ml) was added 2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2- methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (165 mg, 0.38 mmol), DMAP (58 mg, 0.48 mmol), NMM (64 mg, 0.63 mmol) and EDCI (91 mg, 0.48 mmol).
• Step (2) To a solution of 1,2-bis[(Z)-octadec-9-enoxy]pentadecan-3-ol (1.06 g, 1.39 mmol) in THF (20 ml) was added NaH (223 mg, 5.57 mmol). The mixture was stirred at 70°C for 1 hr.2- [2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (1.07 g, 2.09 mmol) was added to this mixture and the mixture was stirred at 70°C for 18 hr.
• the mixture was stirred at 25°C for 18 hr.
• the mixture was dealt with DCM (50 ml), washed with water (50 ml x2), NaCl sat.aq (50 ml) and dried over Na 2 SO 4 .
• reaction was heated at 80°C for 18 hrs.
• reaction mixture was purified through flash chromatography eluted with 10 to 50% ethyl acetate in petroleum ether to give N-[2,3-bis[(Z)-octadec-9- enoxy]propyl]octan-1-amine (4.2 g, 89 % yield) as light yellow oil.
• Step (3) Tert-butyl 3-[2-[2-[2-[2-[tert- butyl(diphenyl)silyl]oxyethoxy]ethoxy]ethoxy]propanoate (0.6 g, 1 mmol) was dissolved in 2:1 DCM-TFA (5 mL) and the solution stirred at room temperature for 1 hour. The resulting mixture was diluted with H 2 O (5 mL) and DCM (10 mL). The solution was stirred vigorously to mix the phases and the solution basified to pH 3 with 2 M NaOH. The layers were separated and the aqueous phase extracted with DCM (30 mL).
• Step (5) To a solution of N-[2,3-bis[(Z)-octadec-9-enoxy]propyl]-3-[2-[2-[2-[2- [tertbutyl(diphenyl)silyl]oxyethoxy]ethoxy]ethoxy]ethoxy]-N-octyl-propanamide (0.42 g, 0.317 mmol) in THF (5 mL) was added Tetra-n-butyl ammonium fluoride (1 M in THF, 0.4 mL) and the mixture was stirred for 1hr at room temperature. TLC indicated that all the starting material was converted into the product.
• the reaction was allowed to stir at room temperature for 48 h.
• the reaction was diluted with dichloromethane and washed with saturated sodium bicarbonate.
• the organic layer was separated and washed with brine, and dried over Na 2 SO 4 .
• the organic layer was filtered and evaporated in vacuo.
• Step (3) To a solution of bis(2,5-dioxopyrrolidin-1-yl) carbonate (2.02 g, 7.88 mmol) in DMF (15 ml) was added 1,2-bis[(Z)- octadec-9-enoxy]pentadecan-3-ol (1.5 g, 1.97 mmol) and 4- Dimethylaminopyridine (963 mg, 7.88mmol). The mixture was stirred at 25°C for 48 hr. The mixture was dealt with EA (50 ml) washed with water (50 xl), LiCl aq (50 mlx2) and dried over Na 2 SO 4 .
• Step (4) To a solution of 2-[2-(2-aminoethoxy)ethoxy]ethanol (2 g, 10.3 mmol) and imidazole (1.62 g, 23.8 mmol) in CH2Cl2 (40 mL) was added TBDPSCl (3.41 g, 12.4 mmol). The reaction was stirred for 18 h at room temperature. The mixture was diluted with CH 2 Cl 2 (30 mL) and washed the resulting mixture with brine. The organic layer was dried over Na 2 SO 4 , filtered and concentrated.
• Step (5) To a solution of 1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyl N-[2-[2-[2-[2- [tertbutyl(diphenyl)silyl]oxyethoxy]ethoxy]ethoxy]ethyl]carbamate (561 mg ,0.46 mmol) in THF (6 ml) was added TBAF (0.92 ml). The mixture was stirred at 25°C for 18 hr.
• Step (6) To a solution of 1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyl N-[2-[2-[2-(2- hydroxyethoxy)ethoxy]ethoxy]ethyl]carbamate (495 mg, 0.5 mmol) in DCM (10 ml) was added 2-[tertbutoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2- methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (329 mg, 0.76 mmol), DIEA (163 mg, 1.26 mmol), DMAP (12 mg) and EDCI (194 mg, 1 mmol).
• Example 22 Synthesis of 2-[[2-[2-[2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9- enoxy]ethyl]tridecoxy]-2-oxoethoxy]ethoxy]ethoxy]ethoxy]-2-oxo- ethyl]amino]ethyl 2-(2- methoxyethylamino)acetate;2,2,2-trifluoroacetic acid (compound (XXV)) p ( ) Synthesis of compound (XXV) Step (1) To a solution of methyl 2-[2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]acetate (1.41 g, 2.7 mmol) in THF/MeOH/Water (4ml/4ml/4ml) was added LiOH ⁇ H 2 O (1.13 g, 27 mmol).
• the mixture was stirred at 25°C for 18 hr.
• the mixture was dealt with EA (50 ml), washed with water (50 ml).
• the aqueous phase was washed with EA (50 mlx2), brine (50 ml) and dried over Na2SO4.
• Step (3) To a solution of 1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyl 2-[2-[2-[2-(2- trityloxyethoxy)ethoxy]ethoxy]ethoxy]acetate (461 mg, 0.37 mmol) in THF/MeOH (5/5 ml) was added Toluene-4-sulfonic acid (461 mg, 0.37 mmol). The mixture was stirred at 25°C for 18 hr. The mixture was dealt with EA (50 ml), washed with NaHCO 3 aq (50 ml x2), brine (50 ml) and dried over Na 2 SO 4 .
• Step (2) In a 250 mL round-bottomed flask was added tert-butyl 3-[2-[2-(2-benzyloxyethoxy) ethoxy]ethoxy]propanoate(3.25 g, 8.38 mmol) in FORMIC ACID (20 ml, 530 mmol) to give a colorless solution. The reaction was heated to 60 °C for 16 hr. The solvent was removed and residue azeotroped with toluene and dried under vacuum to provide 3-[2-[2-(2-benzyloxyethoxy) ethoxy]ethoxy]propanoic acid (2.7 g, 7.35 mmol, 85 % yield) as yellow oil.
• Step (3) A solution of heptadecan-9-yl 8-bromooctanoate (250 mg, 0.542 mmol) in phenylmethanamine (1.2 mL, 10.83 mmol) was allowed to stir at rt for 6 hrs. The reaction was cooled to room temperature and solvents were evaporated in vacuo. The residue was taken-up in ethyl acetate and washed with saturated aqueous sodium bicarbonate.
• Step (4) A solution of heptadecan-9-y18-(benylamino)octanoate (200mg,0.41mmol),nonyl 8-bromooctanoate (172mg, 0.49mmol) and N,N-diisopropylethylamine (100 ⁇ L, 0.57mmol) were dissolved in ACN and was allowed to stir at 62°C for 96h. There action was cooled to rt and solvents were evaporated in vacuo. There residue was taken-up in ethylacetate and washed with saturated aqueous sodium bicarbonate. The organic layer was separated and washed with brine,dried over Na 2 S04 and evaporated in vacuo.
• Step (5) To a solution of nonyl 8-[benzyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (3.94 g,4.95 mmol) in ethyl acetate (50 mL) was added Pd/C (0.79 g) and the mixture was stirred at room temperature under hydrogen for 18 hrs. TLC indicated that the starting material was consumed and one new spot formed. The reaction was filtered through celite and washed with ethyl acetate.
• Step (6) To the solution of nonyl 8-[[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (0.3 g, 0.45 mmol) in dry DCM (10 mL) was added to 3-[2-[2-(2- benzyloxyethoxy)ethoxy]ethoxy]propanoic acid (0.176 g, 0.45 mmol), HATU (0.257 g,0.676 mmol) and DIPEA (0.116 g, 0.901 mmol) then the mixture was stirred at room temperature for 18 hrs. TLC (4% methanol in DCM) indicated that the reaction was finished. The DCM was removed by rotary evaporation.
• Step (8) To a solution of nonyl 8-[3-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]propanoyl-[8-(1- octylnonoxy)-8-oxo-octyl]amino]octanoate (0.802 g,0.921 mmol) and 2-[tert-butoxycarbonyl- [2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (0.801 g,1.84 mmol) in dry DCM (10 mL) was added DMAP (12 mg,0.0921 mmol), DIPEA(0.143 g,1.11 mmol) and then added EDCI(0.212 g,1.11 mmol) at 0 o C.
• Step (9) The mixture of nonyl 8-[3-[2-[2-[2-[tert-butoxycarbonyl-[2-[2-[tert- butoxycarbonyl(2- methoxyethyl)amino]acetyl]oxyethyl]amino]acetyl]oxyethoxy]ethoxy]propanoyl-[8-(1- octylnonoxy)-8-oxo-octyl]amino]octanoate (0.970 g, 0.754 mmol) in 10 mL DCM was added TFA(5 mL) and the mixture was stirred at room temperature for 2 h.
• Step (2) To the solution of 3-benzyloxypropane-1,2-diol (10 g, 0.055 mol) in dry DMF(200 mL) was added NaH (11 g, 0.274 mol) several times at 0oC then the mixture was heated to 80oC for 30 min. Then the reaction was cooled to room temperature and 2-(6- bromohexoxy)tetrahydropyran (36.4 g, 0.137 mol) in dry DMF 100 mL was added under nitrogen and the reaction was heated at 80°C for 18 hrs. TLC indicated that the starting material was consumed. The reaction was quenched with water and extracted with ethyl acetate. The aqueous layer was extracted with ethyl acetate again.
• Step (4) To the solution of 6-[3-benzyloxy-2-(6-hydroxyhexoxy)propoxy]hexan-1-ol(7.0 g, 0.0183 mol) and 2-butyloctanoic acid (14.10 g, 0.0549 mol) in dry dichloromethane (100 mL) were added DIPEA (14.2 g, 0.110 mol), DMAP (0.894 g, 7.32 mmol) and under ice bath added EDCI (9.12 g, 0.0476 mol). The mixture was stirred at room temperature for 18 hrs. The reaction was quenched with NaHCO 3 (100mL) and washed with brine. The organic layer was dried over sodium sulfate, filtered and concentrated.
• Step (7) A mixture of 2,3-bis[6-(2-hexyldecanoyloxy)hexoxy]propanoic acid (2.32 g, 2.96 mmol), EDC HCl (0.85 g, 4.44 mmol), N-Hydroxysuccinimide (0.511 g, 4.44 mmol) in DCM (40 mL). The reaction mixture was stirred for 2 hrs at room temperature. Then N-[2-[2-[2-[2-[2-[2-(2- trityloxyethoxy)ethoxy]ethoxy]ethyl]octan-1-amine (2.10 g, 3.55 mmol) and DIEA (1.15 g, 8.89 mmol) were added.
• Step (8) To a solution of 6-[2-[6-(2-hexyldecanoyloxy)hexoxy]-3-[octyl-[2-[2-[2-[2-(2- trityloxyethoxy)ethoxy]ethoxy]ethyl]amino]-3-oxo-propoxy]hexyl 2-hexyldecanoate (2.1 g, 1.55 mmol) in methanol/THF (40 mL, 1/1 v/v) was added 4-methylbenzenesulfonic acid (1.47 g, 7.44 mmol) in one portion at room temperature and the mixture was stirred at room temperature for 18 hrs.
• Step (9) 6-[2-[6-(2-hexyldecanoyloxy)hexoxy]-3-[2-[2-[2-(2- hydroxyethoxy)ethoxy]ethoxy]ethyl-octyl-amino]-3- oxo-propoxy]hexyl 2- hexyldecanoate (0.4 g, 0.36 mmol) and 2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2- methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (0.24 g, 0.54 mmol) in dry dichloromethane (10 mL) then added DIPEA (0.06 g, 0.43 mmol), DMAP (0.005 g, 0.04 mmol) and added EDCI (0.09 g, 0.43 mmol).
• Step (10) 6-[3-[2-[2-[2-[2-[2-[2-[2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2- methoxyethyl)amino]acetyl]oxyethyl]amino]acetyl]oxyethoxy]ethoxy]ethyl- octyl-amino]-2-[6-(2- hexyldecanoyloxy)hexoxy]-3-oxo-propoxy]hexyl 2-hexyldecanoate (630 mg, 0.411 mmol) in DCM (8 mL) cooled to 0°C was added TFA (1.5 mL) and the mixture was stirred at RT for 4 h.
• Step (2) To a solution of 2,3-bis(oct-7-enoxy)-N-octyl-N-[2-[2-[2-(2- trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]propanamide (8.22 g,9.13 mmol) in ACN/CCl4/H2O (80ml/80ml/80ml) was added NaIO4 (15.6 g ,73 mmol) RUTHENIUM(III) CHLORIDE HYDRATE (412 mg, 1.83 mmol). The mixture was stirred at 25°C for 18 hr.
• the mixture was filtered and dealt with EA (500 ml), washed with Na 2 S 2 O 3 aq (300 ml), brine (300 ml) and dried over Na 2 SO 4 .
• the organic was concentrated and dealt with tert-butyl alcohol/water (120ml/40ml).
• 2-Methyl-2-butene (16 g, 228 mmol) and sodium dihydrogen phosphate (3.29 g, 27.4 mmol) was added to the mixture.
• the mixture was stirred at 25°C for 2 hr.
• Step (3) To a solution of 7-[2-(6-carboxyhexoxy)-3-[octyl-[2-[2-[2-[2-(2- trityloxyethoxy)ethoxy]ethoxy]ethyl]amino]-3- oxo-propoxy]heptanoic acid (5.1 g, 5.45 mmol) in DCM (80 ml) was added (Z)-non-2-en-1-ol (1.86 mg, 13.1 mmol), EDC HCl (3.13 g, 16.3 mmol), DIEA (2.46 g, 19.1 mmol) and DMAP (333 mg). The mixture was stirred at 25°C for 18 hrs.
• Example 27 Synthesis of (Z)-26-(((Z)-octadec-9-en-1-yl)oxy)-5-oxo- 6,9,12,15,18,21,24,28-octaoxa-3-azahexatetracont-37-en-1-yl (2-methoxyethyl)glycinate bis(2,2,2-trifluoroacetate) (compound (XXX)) Compound (XXX) Compound (XXX) was synthesized based on the chemistry shown in the following Scheme.
• Step (1) A mixture of 2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethanol (5.0 g, 17.7 mmol) and N,N-diethylethanamine (3.58 g, 35.4 mmol) in DCM (100 mL) was added [chloro(diphenyl)methyl]benzene (4.94 g, 17.7 mmol). The mixture was stirred for 16 h at room temperature. The mixture was added DCM (100 mL) and washed with water, brine, dried over Na2SO4 and concentrated.
• Step (3) To a mixture of [2-[2-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9- enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy-diphenyl-methyl]benzene (0.7 g, 0.573 mmol) in THF (5 mL)/ MeOH (5 mL) was added Toluene-4-sulfonic acid (0.545 g, 2.86 mmol) and stirred overnight at room temperature. Et3N (1 mL) was added to the reaction mixture and concentrated.
• Step (5) A mixture of 2-[[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9- enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert- butoxycarbonyl-amino]ethyl 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetate (0.6 g, 0.471 mmol) in DCM (5 ml) was added TFA (0.269 g, 2.36 mmol). The mixture was stirred for 3 h at ambient temperature.
• Example 28 Synthesis of 2-[[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9- enoxy]propoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]propyl 2-(2- methoxyethylamino)acetate;2,2,2-trifluoroacetic acid (compound (XXXI)) C Synthesis of compound (XXXI) Step (1) benzyl 2-[tert-butoxycarbonyl-(2-hydroxy-1-methyl-ethyl)amino]acetate In a 250ml round bottom three necked flask equipped with a thermometer and addition funnel, dry Potassium carbonate (9 g, 65.482 mmol) is suspended in acetonitrile (60 mL) under nitrogen.2-aminopropan-1-ol (1.64 g 21.827 mmol) is then added and the mixture cooled to 0°C in
• N-methyl morpholine (1 ml, 9.21 mmol) and 4-(dimethylamino)-pyridine (1.1g, 9.21 mmol) are then added and the mixture cooled to 0°C in an ice bath. Then 1-éthyl-3-(3- diméthylaminopropyl)carbodiimide (1.7 g, 9.21 mmol) is added by portions and the mixture stirred over-night.
• the reaction mixture is the diluted with dichloromethane (30 ml), washed with water (20 ml) and brine (20 ml).
• N-methyl morpholine (0.043 ml, 0.39 mmol) and 4-(dimethylamino)-pyridine (47 mg, 0.39 mmol) are then added and the mixture cooled to 0°C in an ice bath.
• 1-éthyl-3-(3-diméthylaminopropyl)carbodiimide (112 mg, 0.585 mmol) is added by portions and the mixture stirred over-night.
• the reaction mixture is the diluted with dichloromethane (30 ml), washed with water (20 ml) and brine (20 ml).
• the concentrated crude product is purified by flash chromatography on a ISCO Redisep 80 g silica gel column using a Dichloromethane/ methanol (0% to 5% methanol gradient in 35 min.50 ml/min) and then with a dichlorometane/ethyl acetate gradient (0% AcOEt to 100% AcOEt in 35 min.50 ml/min). 240 mg of the desired compound is obtained as an oil.
• Step (3) Ethyl 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]propanoate
• Triethylamine 630 ⁇ L, 4.5mmol
• DMAP 55.9mg – 0.45mmol
• a solution of Boc2O 1.209g – 5.43mmol in Dichloromethane (3 mL) is then added over a 10min period.
• Step (3) 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]propanoic acid
• ethyl 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]propanoate (198mg, 0.72mmol) in Methanol (5.4mL) cooled to 0°C is added a 2M solution of Lithium Hydroxide Hydrate in Water (1.8mL – 3.6mmol).
• the solution is stirred 30min at 0°C and 7h at room temperature.
• the pH is then decreased to 3-4 by addition of DOWEX 50W X8 acidic resin.
• the resin is filtrated and the filtrate partially evaporated under reduced pressure.
• Example 30 Lipid Nanoparticles (LNPs) preparation Preparation of the organic solvent Lipids were dissolved in ethanol at molar ratios of 50/10/38.5/1.5 or 35/2.5/46.5/16 (ionizable lipid:neutral lipid:cholesterol:PEGylated lipid) PEGylated lipid may be PEG2000-PE or DMG-PEG2000. Neutral lipid may be DPSC or DOPE. Two ionizable cationic lipidic compounds were used to manufacture the LNPs: L319 (DLin-MC3-DMA) and lipidic compound of formula (III) (or DOG-CLEAVE).
• LNPs Lipid Nanoparticles
• LNPs 319) Preparation of 1.5 mL of organic solvent for L319-containing LNPs (LNPs 319) formulation 3.9 mg of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine - Avanti Polar Lipids: 850365), 2 mg of PEG2000-PE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (ammonium salt) - Avanti Polar Lipids: 880150P) and 7.4 mg of cholesterol (Sigma Aldrich: C3045) were dissolved with 1319 ⁇ L of ethanol.
• DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine - Avanti Polar Lipids: 850365
• PEG2000-PE 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (
• L319 stock solution 100mg/mL in ethanol - DLin-MC3-DMA - Maier et al., Molecular Therapy, 2013, 21 (8): 1570–15778 was added to obtain 20 mg/mL of lipid phase solution.
• PEG2000-PE was replaced with DMG-PEG.
• aqueous solvent Preparation of the aqueous solvent Preparation of 1.8 mL of aqueous phase for LNPs 319
• a non-replicative mRNA encoding A/Netherlands HA (SEQ ID NO: 1) was used.
• the influenza HA mRNA was produced by in vitro transcription (IVT) as an unmodified mRNA transcript from a linear DNA template generated by PCR, using wild type bases and T7 RNA polymerase (Avci-Adali et al (J. Vis. Exp. (93), e51943, doi:10.3791/51943 (2014) and Kwon et al., Biomaterials 156 (2016) 172e193).
• mRNA concentration was calculated to be 305 ⁇ g/mL.
• the mRNA solution was prepared in 50 mM citrate buffer pH 4.0. Preparation of 1.8 mL of aqueous phase for LNPs Lip.
• (III) lipidic compound of formula (III)-containing LNPs
• LNPs preparation LNPs were prepared using a NanoAssemblR equipment according to manufacturer recommendations.
• the aqueous and organic phases were each loaded in a syringe suitable for NanoAssemblR according to manufacturer recommendations.
• the flow rate was set up at a ratio: 3:1 and total flow rate: 4ml/min.
• the aqueous and lipid phases were then mixed to obtain the LNPs.
• LNPs L319 purification and harvest The obtained LNPs were dialyzed against a citrate buffer (50mM - pH 4.0) to remove residual ethanol and then twice against a PBS buffer (pH 7.4). Each dialysis was carried out at least during 12 hours at 4°C.
• the LNPs were then filtered through a 0.22 ⁇ m filter and store under nitrogen at +4°C. LNPs Lip.
• the obtained LNPs were dialyzed against a citrate buffer (50mM - pH 4.0) to remove residual ethanol and then twice against a citrate buffer (10 mM - pH 6.3) containing sucrose (8%). Each dialysis was carried out at least during 12 hours at 4°C. The LNPs were then filtered through a 0.22 ⁇ m filter and store under nitrogen at +4°C. The dialysis against an aqueous solvent at pH 6.3 allowed the hydrolysis and self- rearrangement of the ionizable lipid as disclosed herein into a neutral lipid and the removal of the cationic polar head. The zeta potential of the obtained LNPs is therefore closed to 0 mV.
• Example 31 Characterization of LNPs RNA titration / encapsulation rate
• the percentage of encapsulated mRNA and concentration of mRNA in LNPs were measured using the Quant-iT Ribogreen RNA reagent kit according to manufacturer recommendations (Invitrogen Detection Technologies) and quantified with a fluorescent microplate reader or a standard spectrophotometer using fluorescein excitation and emission wavelength.
• LNPs were diluted in Tris/EDTA assay buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5).
• RNA samples were diluted in Tris/EDTA assay buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) containing 0.5 %(v/v) Triton X100.
• Ribogreen dye 200X diluted was added to the samples (50/50 mix; sample/Ribogreen reagent), mixed thoroughly and incubated 5 min at room temperature in the dark. Fluorescence was measured on the plate reader. Lipid quantification It was assumed that there was no loss of lipids during the formulation process. The total lipid concentration before dialysis was then assumed to be 5 mg/mL. The final lipid concentration was defined by taking into account the dilution factor occurring during the dialysis step.
• Particle size distribution, polydispersity index Zeta potential, and osmolarity, Zeta potential and particle size distribution of LNPs were measured by using a zeta sizer Nano ZS light scattering instrument (Malvern Instruments) according to manufacturer recommendations. Particle sizes were reported as the Z-average size (harmonic intensity averaged particle diameter) along with the Polydispersity Index (PDI), an indicator of the “broadness” of the particle size distribution. Samples were diluted to 1/100 in phosphate buffered saline (PBS) before measurement.
• PBS phosphate buffered saline
• Example 32 immunogenicity of LNPs comprising influenza HA mRNA in mice
• the aim of the study was to compare the immunogenicity of different doses of natural, non-replicative mRNA encoding full-length hemagglutinin (HA) of influenza virus strain A/Netherlands/602/2009 (H1N1) formulated in 2 different lipid nanoparticles, LNPs L319 and LNPs (III)/DOG-CLEAVE.
• the LNPs were prepared as described in Example 30.
• the LNPs contain lipids in the following molar ratios of 50/10/38.5/1.5 (ionizable cationic lipid/neutral lipid/Chol/PEG-ylated lipid).
• mice The ionizable lipids were formulated with DSPC/Chol/PEG-PE.
• Mouse immunization procedure 2 groups of 8 BALBc/ByJ mice (8 week-old at D0) received two intramuscular (IM) injections, given three weeks apart (D0 and D21), of 5 ⁇ g of mRNA with each the 2 LNP formulations. The L319 was used as benchmark.
• IM intramuscular
• mice were immunized with PBS buffer and as positive control group, 8 mice received 10 ⁇ g of monovalent Flu vaccine A/California/07/2009 (H1N1) strain derived from VaxigripTM, according to the same immunization schedule.
• H1N1 monovalent Flu vaccine A/California/07/2009
• HI titers hemagglutination inhibiting antibody titers
• each serum was treated with a receptor-destroying enzyme (RDE) (neuraminidase from Vibrio cholerae – Type III - Sigma Aldrich N7885) and with chicken red blood cells (cRBCs). Briefly, 10 mU/mL of RDE was added to each serum. The mix was then incubated 18 h at 37°C, followed by 1 h inactivation at 56°C. To cool, the mixture “serum-RDE” was placed in a time range from 30 min to 4 hours at 4°C.
• RDE receptor-destroying enzyme
• the "serum-RDE" mixture was then absorbed on 10% cRBCs in PBS for 30 min, at room temperature, and then centrifuged at 5°C, 10 min at 700 g. The supernatant corresponding to 10-fold diluted serum was collected to perform the HI assay.
• Chicken red blood cells (0.5% in PBS) (50 ⁇ L) were then added to each well and inhibition of hemagglutination or hemagglutination was visually read after one hour at room temperature.
• the titer in HI antibody is the reciprocal of the last dilution giving no hemagglutination. A value of 5 corresponding to half of the initial dilution (1:10) was arbitrary given to all sera determined negative in order to perform statistical analysis.
• mice The antibody responses elicited against A/California/7/2009 (H1N1) were measured by HI assay in individual sera collected from all animals at D21 and D42.
• Mean HI titers measured in mice after one and two IM injections of LNPs Results analyzed according the total mRNA content per dose showed the following: Following two injections with the LNPs, high mean HI titers was observed. After the second injection of mRNA (post-2 immunization (D42)), animals administered with mRNA/LNPs (III) showed detectable HI responses as observed with mRNA/L319 LNPs.
• mice which received 5 ⁇ g of total mRNA in LNP L319 at the injection site on D22, D23 or D24, i.e.1 to 3 days following the booster injection.
• LNPs reagents 1,3-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG) were purchased from Avanti Polar Lipids (Alabaster, Albany).
• 3 ⁇ -Hydroxy-5- cholestene, 5-Cholesten-3 ⁇ -ol (Cholesterol) and Acrodisc Syringe Filters with Supor Membrane, Sterile-0.2 ⁇ m, 13mm, were purchased Sigma Aldrich (St. Louis, Missouri).
• Coatsome SS-OP was purchased from NOF America Corporation (White Plains, NY headquartered in Japan). Nuclease-free water, 10X PBS Buffer pH 7.4, Sodium Citrate, Dihydrate, Citric Acid, Sodium Chloride, Sucrose, Ethyl Alcohol, BD Vacutainer General Use Syringe Needles (BD Blunt Fill Needle 18G), BD Slip Tip Sterile Syringes (3 ml & 1 mL), Amicon Ultra Centifugal Filter Units, DNA-free microcentrifuge tubes (1.5mL), Invitrogen RNase-free Microfuge Tubes (0.5mL), Invitrogen Conical Tubes (15 mL) (DNase-RNase-free), Fisher Brand Semi-Micro Cuvette, Quant-iTTM RiboGreen® RNA Assay Kit and RnaseZapTM were purchased from Thermo Fisher Scientific (Waltham, Massachusetts).
• mRNA encoding luciferase was purchased from TriLinkTM Biotechnologies (mRNA-Luc - Ref.: L-7602). All LNPs were manufactured using the NanoAssemblr Benchtop from Precision Nanosystems (British Columbia, Canada).
• LNPs manufacturing The following buffer systems were used: - Phosphate Buffer Saline (PBS): 8 mM Na 2 HPO 4 , and 2 mM KH 2 PO 4 , 137 mM NaCl, 2.7 mM KCl, pH 7.4 - Citrate Buffer-10 mM: 5 mM Sodium citrate, 5 mM citric acid, 150 mM Sodium Chloride, pH 4.5 - Citrate Buffer-50 mM: 25 mM Sodium citrate, 25 mM citric acid, pH 4.0 - Citrate-Sucrose Buffer: 5 mM Sodium citrate, 5 mM citric acid, 8% (w/v) sucrose, pH 6.3
• PBS Phosphate Buffer Saline
• total lipid SS lipid + DOPC + Chol
• TABLE 3 LNP mix working solution for LNP SS-OP L S D C D E
• T LNPs (III): lipidic compound (III)/DSPC/Chol/DMG-PEG 50/10/38.5/1.5 533 ⁇ L of mixed lipids solution (total lipid concentration 10 mM) was prepared in a 1.5 mL conical tube by mixing the following solutions:
• TABLE 4 LNP mix working solution for LNPs (III) Aqueous nucleic acid containing phase The following mRNA containing aqueous solutions were used for manufacturing the LNPs.
• the LNPs were manufactured with the NanoAssemblrTM according to manufacturer’s recommendations the following parameters: TABLE 6: NanoAssemblrTM parameters Vo Flo To Le Rig Sta En The LNPs were formulated to encapsulate 4.25 ⁇ g of mRNA (final content) LNPs harvest and purification The harvested LNPs SS-OP were three-time washed by dilution in PBS and filtration (100KD MWCO). The resuspended LNPs were then filtered on 0.2 ⁇ m filter.
• the harvested LNPs (III) were three-time washed by dilution in 10 mM citrate / 8% sucrose buffer, pH 6.3 and filtration (100KD MWCO). The resuspended LNPs were then filtered on 0.2 ⁇ m filter. Animals study 2 groups, each of 4 of SKH1 hairless mice, female 10-12 weeks old, were tested with LNPs SS-OP and LNPs Lip. (III) encapsulating a luciferase encoding mRNA. A last group of 2 mice was injected with PBS and used as control. The LNPs were formulated to encapsulate 4.25 ⁇ g of mRNA (final content).
• the organ data is summarized in Table 7. Significant transduction and luciferase expression were observed in the spleen for LNPs (III) (200-folds higher) compared to other organs (spleen, kidney, heart and lungs).
• the specificity of LNPs (III) targeting spleen compared to liver was 43 folds higher than the LNPs SS-OP (Table 8).
• the LNPs SS-OP although demonstrated higher absolute expression in the spleen compared to LNPs (III), the relative expression in the liver was significantly higher showing that the LNPs SS-OP formulation is better suited for targeting liver than spleen.
• LNPs (III) can be effectively used for delivery of the nucleic acid specifically to the spleen via intravenous administration.
• TABLE 8 Radiance per organ
• TABLE 9 Distribution of LNPs to other organs with respect to liver
• Example 34 BioImaging following IM injection The purpose of the study was to determine in vivo protein expression after injection of LNP formulations in normal mice. Materials and methods: Ten weeks-old female BALB/c ByJ mice were obtained from Charles River lab (Les Oncins, Saint-Germain-Nuelles, 69210, France).
• mice were injected by intramuscular (IM) route with 50 ⁇ l of LNPs 319 or LNPs (III) (prepared as described in Example 30) containing 5 ⁇ g of mRNA encoding luciferase (mRNA-Luc - Ref.: L-7602 TriLinkTM Biotechnologies).
• Luciferin potassium salt D-luciferin, K+ salt Fluoprobes, Interchim
• Optical imaging was performed using the IVIS Spectrum CT device (PerkinElmer Inc., Paris, France). Bioluminescence acquisition was initiated 15 min after the injection of the substrate.
• Example 35 Formulation of hEPO mRNA in LNPs comprising cleavable lipids of the invention
• Lipid nanoparticles CleanCap® EPO mRNA (5moU), a non-replicative, highly purified, mRNA encoding the human erythropoietin was obtained from TriLink Biotechnologies, San Diego, CA (catalogue number L7209; hEPO mRNA). This mRNA is capped using CleanCap, TriLink's proprietary co-transcriptional capping method, which results in the naturally occurring Cap 1 structure with high capping efficiency. It is polyadenylated, modified with 5-methoxyuridine and optimized for mammalian systems. It mimics a fully processed mature mRNA.
• LNPs were prepared at a concentration of 60 ⁇ g of hEPO mRNA/mL in PBS 1X. Intramuscular injection of mice with LNPs Lip. (III), or LNPs with one of the compounds or one of the compounds (VI), (XVII), (XIX), (XXI), (XXII), (XXIV), (XXVII) and (XXVIII), containing hEPO mRNA and detection of hEPO expression in the serum was thereafter carried out.
• Animals Female Balb/c ByJ mice (7 weeks of age at receipt) were purchased from Charles River Laboratories (Saint-Germain-Nuelles, France) and housed for one-week acclimation before starting the study. Mice were identified individually by fur coloration.
• Blood Samples Blood samples were collected 6 hours post-injection by carotid section under deep anesthesia with Imal relie/Rompun (1.6 mg of Ketamine / 0.32 mg of Xylazine) in serum- separation tubes (BD Vacutainer # BD367957). Sera were aliquoted and stored at -20°C until hEPO determination. hEPO determination in mouse serum hEPO expression in mouse sera was assessed using human Erythropoietin Quantikine IVD ELISA kit (R&D Systems #DEP00). The ELISA was performed following supplier’s instructions. Briefly, sera were added in pre-coated plates and incubated for one hour at room temperature under orbital shaking.
• an UHPLC-CAD-MS method was used to analyze the lipid constituents at different steps of the LNP preparation process described in Example 30, i.e., in the starting ethanol solution, after the first dialysis step and in the final LNP.
• UHPLC-CAD-MS method for LNP lipid content and integrity analysis For the separation and analysis of the different lipid components in LNPs, a RP- UHPLC method with Charged Aerosol Detection (CAD) and mass spectrometry detection (MS) was used. The equipment and chromatographic conditions are described in the tables below.
• Results are shown in the table below showing the relative amounts of DOG-Cleave, DOG-Cleave transient and DOG-OH during the LNP formulation process.
• TABLE 13 Relative amounts of DOG-Cleave (III), DOG-Cleave transient and DOG- OH during the LNP formulation process.
• the UHPLC-CAD-MS analysis confirm the cleavage of lipid compound of formula (III) (DOG-Cleave) and its transformation into DOG-OH during the LNP formulation process.
• Example 38 Immunogenicity of LNPs comprising influenza HA mRNA in mice The aim of the study was to evaluate the immunogenicity induced with different LNPs made with different lipidic compounds as disclosed herein and containing non-replicative mRNA encoding full-length hemagglutinin (HA) of influenza virus.
• HA hemagglutinin
• BALBc/ByJ mice (8 weeks old at D0; 8 per group) were immunized as described in Example 32 with 5 ⁇ g of natural, non-replicative mRNA encoding full-length hemagglutinin (HA) of influenza virus strain A/Netherlands/602/2009 (H1N1) formulated in 4 different lipid nanoparticles, LNPs L319, LNPs (III) [DOG-Cleave], LNPs (XXI), and LNPs (XIX).
• LNPs were prepared as described in Example 31 and were always composed of ionizable or cleavable lipid/DSPC/Chol/DMG-PEG2000 at a 50:10:38.5:1.5 molar ratio. Ratio N/P was 6 for LNPs L319 and N/P was 12 for the cleavable lipid-containing LNPs.
• HI titers were measured 3 weeks following the second immunization as described in Example 32 and reported on Figure 15.

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